pIVT-dCas9-EGFP

LOCUS       pIVT-dCas9-EGFP               7891 bp ds-DNA     circular SYN 22-SEP-2021
DEFINITION  in vitro transcription template for dCas9-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pIVT-dCas9-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7891)
  TITLE     Parallel pathways for recruiting effector proteins determine 
            centromere drive and suppression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7891)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 22, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7891
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             201..1061
                     /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     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2144..2174
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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)."
     primer_bind     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2246..2264
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2246..2263
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2276..2295
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2276..2294
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           2337..2379
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      2437..2446
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2443..6546
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6562..7278
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6607..6628)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6868..6887)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7215..7236
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     3'UTR           7309..7434
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(7497..7514)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7497..7513)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7506..7528)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7722..7741)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7841..7863
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctat cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctc caacgattta ggtgacacta tagaagagaa ggaattaata
     2281 cgactcacta tagggagaga gagagaatta ccctcactaa agggaggaga agcttgcttg
     2341 ttctttttgc agaagctcag aataaacgct caactttggc agatcaagct tgcatgcctg
     2401 caggtcgact atagggagac ccaagcttgg cgcgccgcca ccatggacaa gaagtactcc
     2461 attgggctcg ctatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag
     2521 gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac
     2581 ctcattggcg ccctcctgtt cgactccggg gagacggccg aagccacgcg gctcaaaaga
     2641 acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt
     2701 agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg
     2761 gtggaggagg ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg
     2821 gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact
     2881 gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga
     2941 cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc
     3001 caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt
     3061 gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc
     3121 gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtcactc
     3181 gggctgaccc ccaactttaa atctaacttc gacctggccg aagatgccaa gcttcaactg
     3241 agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac
     3301 gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg
     3361 cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa gcgctatgat
     3421 gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag
     3481 tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga
     3541 gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc
     3601 gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac
     3661 aatggaagca tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa
     3721 gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt
     3781 cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact
     3841 cgcaaatcag aagagaccat cactccctgg aacttcgagg aagtcgtgga taagggggcc
     3901 tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag
     3961 gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag
     4021 gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga gcagaagaaa
     4081 gctatcgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa
     4141 gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc
     4201 ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc
     4261 ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt
     4321 gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa
     4381 gtcatgaaac agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg
     4441 atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat
     4501 ggatttgcca acaggaactt catgcagttg atccatgatg actctctcac ctttaaggag
     4561 gacatccaga aagcacaagt ttctggccag ggggacagtc ttcacgagca catcgctaat
     4621 cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa
     4681 ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag
     4741 aaccaaacta cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag
     4801 ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt
     4861 cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag
     4921 gaactggaca tcaatcggct ctccgactac gacgtggatg ctatcgtgcc ccagtctttt
     4981 ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaaaa tagagggaag
     5041 agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg
     5101 ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt
     5161 ggcctgtctg agttggataa agccggcttc atcaaaaggc agcttgttga gacacgccag
     5221 atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat
     5281 gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc
     5341 agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat
     5401 gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct
     5461 gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag
     5521 caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc
     5581 aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac
     5641 ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc
     5701 ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggcttctcc
     5761 aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg
     5821 gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg
     5881 gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc
     5941 acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga
     6001 tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt
     6061 gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg taacgagctg
     6121 gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa
     6181 gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt
     6241 gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac
     6301 ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca
     6361 gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac
     6421 ttcgacacca ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca
     6481 ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt
     6541 ggagacgcta gcggctctag aatggtgagc aagggcgagg agctgttcac cggggtggtg
     6601 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     6661 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     6721 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     6781 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     6841 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     6901 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     6961 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     7021 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     7081 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     7141 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     7201 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     7261 atggacgagc tgtacaagta gtgactgact aggatctggt taccactaaa ccagcctcaa
     7321 gaacacccga atggagtctc taagctacat aataccaact tacactttac aaaatgttgt
     7381 cccccaaaat gtagccattc gtatctgctc ctaataaaaa gaaagtttct tcacattcta
     7441 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa ancccccccc ccccaagggc gaattcactg
     7501 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     7561 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     7621 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     7681 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     7741 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     7801 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     7861 aggttttcac cgtcatcacc gaaacgcgcg a
//