PB-NEO-GFP-RFP

LOCUS       PB-NEO-GFP-RFP                7599 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  seamless gene editing and component of Piggybac transposase system.
ACCESSION   .
VERSION     .
KEYWORDS    PB-NEO-GFP-RFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7599)
  TITLE     Bi Lab mammalian expression constructs
REFERENCE   2  (bases 1 to 7599)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7599
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   440..474
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    complement(768..801)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     enhancer        868..1171
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1172..1375
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             1404..2195
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1458..1477)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2068..2087
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             2217..2282
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             2289..3008
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2289..3005
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2334..2355)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2595..2614)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2942..2963
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3131..3212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(3219..3252)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     repeat_region   3443..3505
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        3594..3897
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3898..4101
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      4128..4137
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4134..4811
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Strongin et al., 2007)"
                     /label=DsRed-Monomer
                     /note="mammalian codon-optimized"
                     /translation="MDNTEDVIKEFMQFKVRMEGSVNGHYFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYMKLSFPEGFTWERSMNFEDGGV
                     VEVQQDSSLQDGTFIYKVKFKGVNFPADGPVMQKKTAGWEPSTEKLYPQDGVLKGEISH
                     ALKLKDGGHYTCDFKTVYKAKKPVQLPGNHYVDSKLDITNHNEDYTVVEQYEHAEARHS
                     GSQ"
     primer_bind     complement(4314..4334)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4532..4551
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4934..5055
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5025..5044
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5199..5220)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5378..5394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5378..5394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5391..5413)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5402..5418
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5426..5456)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5471..5492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5609..5626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5780..6368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5860..5879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6539..7399)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7162..7181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7400..7504)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7572..7590
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggc gcgcctcatg
      421 attaattaaa tcgatttaac cctagaaaga tagtctgcgt aaaattgacg catgcattct
      481 tgaaatattg ctctctcttt ctaaatagcg cgaatccgtc gctgtgcatt taggacatct
      541 cagtcgccgc ttggagctcc cgtgaggcgt gcttgtcaat gcggtaagtg tcactgattt
      601 tgaactataa cgaccgcgtg agtcaaaatg acgcatgatt atcttttacg tgacttttaa
      661 gatttaactc atacgataat tatattgtta tttcatgttc tacttacgtg ataacttatt
      721 atatatatat tttcttgtta tagatatcat cgatgcgatc gcagctcata acttcgtata
      781 atgtatgcta tacgaagtta tctcgactag ttattaatag taatcaatta cggggtcatt
      841 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      901 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      961 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     1021 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     1081 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     1141 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     1201 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     1261 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     1321 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt
     1381 agtgaaccgt cagatccgct agcatgattg aacaagatgg attgcacgca ggttctccgg
     1441 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     1501 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     1561 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     1621 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     1681 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     1741 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     1801 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     1861 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     1921 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     1981 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     2041 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     2101 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     2161 gcatcgcctt ctatcgcctt cttgacgagt tcttcctcga gaaaattgtc gctcctgtca
     2221 aacaaactct taactttgat ttactcaaac tggctgggga tgtagaaagc aatccaggtc
     2281 caggatccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2341 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2401 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2461 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     2521 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2581 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2641 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2701 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2761 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2821 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2881 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     2941 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     3001 acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
     3061 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     3121 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3181 aaatttcaca aataaagcat ttttttcact gcagatccat aacttcgtat aatgtatgct
     3241 atacgaagtt atcgacctgc aggcatgcgt cgactttgtt actttataga agaaattttg
     3301 agtttttgtt tttttttaat aaataaataa acataaataa attgtttgtt gaatttatta
     3361 ttagtatgta agtgtaaata taataaaact taatatctat tcaaattaat aaataaacct
     3421 cgatatacag accgataaaa cacatgcgtc aattttacgc atgattatct ttaacgtacg
     3481 tcacaatatg attatctttc tagggttaac ctaggaccgg tggtaccaag ctttagttat
     3541 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     3601 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     3661 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     3721 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     3781 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     3841 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     3901 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     3961 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     4021 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     4081 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgagcgcc accatggaca
     4141 acaccgagga cgtcatcaag gagttcatgc agttcaaggt gcgcatggag ggctccgtga
     4201 acggccacta cttcgagatc gagggcgagg gcgagggcaa gccctacgag ggcacccaga
     4261 ccgccaagct gcaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
     4321 cccagttcca gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactaca
     4381 tgaagctgtc cttccccgag ggcttcacct gggagcgctc catgaacttc gaggacggcg
     4441 gcgtggtgga ggtgcagcag gactcctccc tgcaggacgg caccttcatc tacaaggtga
     4501 agttcaaggg cgtgaacttc cccgccgacg gccccgtaat gcagaagaag actgccggct
     4561 gggagccctc caccgagaag ctgtaccccc aggacggcgt gctgaagggc gagatctccc
     4621 acgccctgaa gctgaaggac ggcggccact acacctgcga cttcaagacc gtgtacaagg
     4681 ccaagaagcc cgtgcagctg cccggcaacc actacgtgga ctccaagctg gacatcacca
     4741 accacaacga ggactacacc gtggtggagc agtacgagca cgccgaggcc cgccactccg
     4801 gctcccagta gagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt
     4861 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     4921 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4981 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     5041 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     5101 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     5161 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     5221 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     5281 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     5341 atcggaaccc taaagggaga agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5401 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5461 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5521 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5581 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5941 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6181 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6301 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6361 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6541 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6601 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6661 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6721 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6781 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6841 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6901 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6961 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7021 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7081 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7141 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7201 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7261 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7321 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7381 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7441 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7501 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     7561 taacctataa aaataggcgt atcacgaggc cctttcgtc
//