PB_rtTA_BsmBI

LOCUS       PB_rtTA_BsmBI                8652 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  PiggyBac cargo vector expressing rtTA, contains BsmBI sites for 
            sgRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    PB_rtTA_BsmBI
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8652)
  AUTHORS   Schertzer MD, Thulson E, Braceros KCA, Lee DM, Hinkle ER, Murphy RM,
            Kim SO, Vitucci EM, Calabrese JM
  TITLE     A piggyBac-based toolkit for inducible genome editing in mammalian 
            cells.
  JOURNAL   RNA. 2019 May 17. pii: rna.068932.118. doi: 10.1261/rna.068932.118.
  PUBMED    31101683
REFERENCE   2  (bases 1 to 8652)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..8652
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_RNA        266..341
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        372..1583
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     1420..1439
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             1642..2388
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    2426..2997
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2591..2608)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2818..2837
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3013..3807
                     /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(3067..3086)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3677..3696
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3849..3970
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3886..3905)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3940..3959
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4733..4767
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5258..5274)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5258..5274)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5271..5293)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5282..5298
                     /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(5306..5336)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5351..5372
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5489..5506)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5660..6248)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5740..5759)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6419..7279)
                     /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     7042..7061
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7280..7384)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7410..7865)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7547..7568)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7759..7778
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7992..8014
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8006..8023
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8007..8023
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   8153..8215
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttatcg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gagacggacg tctctgtttt agagctagaa atagcaagtt aaaataaggc
      301 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttcc gcattaaccc
      361 acccaagatc tggcctccgc gccgggtttt ggcgcctccc gcgggcgccc ccctcctcac
      421 ggcgagcgct gccacgtcag acgaagggcg cagcgagcgt cctgatcctt ccgcccggac
      481 gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa
      541 ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg
      601 aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg
      661 tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc
      721 gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg
      781 gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt
      841 ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt
      901 tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag
      961 gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt
     1021 cttgaggcct tcgctaatgc gggaaagctc ttattcgggt gagatgggct ggggcaccat
     1081 ctggggaccc tgacgtgaag tttgtcactg actggagaac tcggtttgtc gtctgttgcg
     1141 ggggcggcag ttatggcggt gccgttgggc agtgcacccg tacctttggg agcgcgcgcc
     1201 ctcgtcgtgt cgtgacgtca cccgttctgt tggcttataa tgcagggtgg ggccacctgc
     1261 cggtaggtgt gcggtaggct tttctccgtc gcaggacgca gggttcgggc ctagggtagg
     1321 ctctcctgaa tcgacaggcg ccggacctct ggtgagggga gggataagtg aggcgtcagt
     1381 ttctttggtc ggttttatgt acctatcttc ttaagtagct gaagctccgg ttttgaacta
     1441 tgcgctcggg gttggcgagt gtgttttgtg aagtttttta ggcacctttt gaaatgtaat
     1501 catttgggtc aatatgtaat tttcagtgtt agactagtaa attgtccgct aaattctggc
     1561 cgtttttggc ttttttgtta gacgaagctt ggtaccgagc tcggatctcc accccgtacc
     1621 ggtcctgcag tcgaattcac catgtctaga ctggacaaga gcaaagtcat aaacggagct
     1681 ctggaattac tcaatggtgt cggtatcgaa ggcctgacga caaggaaact cgctcaaaag
     1741 ctgggagttg agcagcctac cctgtactgg cacgtgaaga acaagcgggc cctgctcgat
     1801 gccctgccaa tcgagatgct ggacaggcat catacccact tctgccccct ggaaggcgag
     1861 tcatggcaag actttctgcg gaacaacgcc aagtcatacc gctgtgctct cctctcacat
     1921 cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag agaaacagta cgaaaccctg
     1981 gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc actgtacgct
     2041 ctgtccgccg tgggccactt tacactgggc tgcgtattgg aggaacagga gcatcaagta
     2101 gcaaaagagg aaagagagac acctaccacc gattctatgc ccccacttct gagacaagca
     2161 attgagctgt tcgaccggca gggagccgaa cctgccttcc ttttcggcct ggaactaatc
     2221 atatgtggcc tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac
     2281 gattttgact tagacatgct cccagccgat gcccttgacg actttgacct tgatatgctg
     2341 cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccgggtaact aagtaaggat
     2401 ccgcggccgc actagaggaa ttccgcccct ctcccccccc cccctaacgt tactggccga
     2461 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     2521 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     2581 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     2641 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     2701 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     2761 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     2821 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     2881 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
     2941 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataagct
     3001 taccggtcca ccatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3061 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3121 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3181 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3241 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3301 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3361 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3421 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3481 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3541 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3601 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3661 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3721 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3781 tatcgccttc ttgacgagtt cttctgacaa tcaacctctg gattacaaaa tttgtggtta
     3841 actaactaaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3901 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3961 atgtatctta tcatgtctgg aattgactca aatgatgtca attagtctat cagaagctca
     4021 tctggtctcc cttccggggg acaagacatc cctgtttaat atttaaacag cagtgttccc
     4081 aaactgggtt cttatatccc ttgctctggt caaccaggtt gcagggtttc ctgtcctcac
     4141 aggaacgaag tccctaaaga aacagtggca gccaggttta gccccggaat tgactggatt
     4201 ccttttttag ggcccattgg tatggctttt tccccgtatc cccccaggtg tctgcaggct
     4261 caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg
     4321 gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc
     4381 ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt
     4441 tggggggggg ctgtccctga tatctataac aagaaaatat atatataata agttatcacg
     4501 taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta
     4561 aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact
     4621 taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg
     4681 cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc
     4741 aattttacgc agactatctt tctagggtta atctagctgc atcaggatca tatcgtcggg
     4801 tcttttttcc ggctcagtca tcgcccaagc tggcgctatc tgggcatcgg ggaggaagaa
     4861 gcccgtgcct tttcccgcga ggttgaagcg gcatggaaag agtttgccga ggatgactgc
     4921 tgctgcattg acgttgagcg aaaacgcacg tttaccatga tgattcggga aggtgtggcc
     4981 atgcacgcct ttaacggtga actgttcgtt caggccacct gggataccag ttcgtcgcgg
     5041 cttttccgga cacagttccg gatggtcagc ccgaagcgca tcagcaaccc gaacaatacc
     5101 ggcgacagcc ggaactgccg tgccggtgtg cagattaatg acagcggtgc ggcgctggga
     5161 tattacgtca gcgaggacgg gtatcctggc tggatgccgc agaaatggac atggataccc
     5221 cgtgagttac ccggcgggcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5281 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5341 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5401 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5461 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5521 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5581 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5641 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5701 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5761 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5821 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5881 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5941 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6001 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6061 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6121 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6181 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6241 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6301 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6361 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6421 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6481 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6541 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6601 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6661 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6721 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6781 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6841 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6901 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6961 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7021 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7081 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7141 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7201 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7261 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7321 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7381 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     7441 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     7501 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     7561 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     7621 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     7681 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     7741 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     7801 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     7861 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     7921 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     7981 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cctcgttcat
     8041 tcacgttttt gaacccgtgg aggacgggca gactcgcggt gcaaatgtgt tttacagcgt
     8101 gatggagcag atgaagatgc tcgacacgct gcagaacacg cagctagatt aaccctagaa
     8161 agataatcat attgtgacgt acgttaaaga taatcatgtg taaaattgac gcatgtgttt
     8221 tatcggtctg tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat
     8281 ttacacttac atactaataa taaattcaac aaacaattta tttatgttta tttatttatt
     8341 aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttat gagggacagc
     8401 ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga
     8461 gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc
     8521 ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc
     8581 tgctctttga gcctgcagac acctgggggg atacggggaa aaggcctcca cggccccata
     8641 aagatggtta at
//