LOCUS       sequence-313940-        7519 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses copGFP and puromycin resistance genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7519)
  AUTHORS   Wang B, Wang Z, Wang D, Zhang B, Ong SG, Li M, Yu W, Wang Y
  TITLE     krCRISPR: an easy and efficient strategy for generating conditional 
            knockout of essential genes in cells.
  JOURNAL   J Biol Eng. 2019 Apr 24;13:35. doi: 10.1186/s13036-019-0150-y. 
            eCollection 2019.
  PUBMED    31049076
REFERENCE   2  (bases 1 to 7519)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Eng."; date: "2019-04-24"; volume: "13:35. doi"; pages: " 
            10.1186/s13036-019-0150-y. eCollection 2019"
COMMENT     Sequence Label: rescue plasmid
FEATURES             Location/Qualifiers
     source          1..7519
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..315
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    9..27
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    45..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    80..98
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    116..134
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    152..170
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    187..205
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    223..241
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     288..312
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             425..478
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             509..1171
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
                     FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
                     EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
                     YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
     CDS             1256..1312
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1313..1909
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1313..1332)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1809..1829
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1916..2911
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     polyA_signal    2931..3065
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2955..2974)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3009..3028
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3208..3230)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5430..5534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5535..5603,5604..6395)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5753..5772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6566..7154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7055..7074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(7343..7363)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     protein_bind    7420..7438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7456..7474
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7492..7510
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag
       61 agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
      121 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg
      181 agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
      241 aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
      301 aaccgtcaga tcgcctggag aattcgagct cggtacccgg ggatcctcta gtcagctgac
      361 gcgtgctagc gcggccgcat cgataagctt gtcgacgata tctctagagc gatcgctgac
      421 cggtgagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggcccttc
      481 cggaatggag agcgacgaga gcggcctgcc cgccatggag atcgagtgcc gcatcaccgg
      541 caccctgaac ggcgtggagt tcgagctggt gggcggcgga gagggcaccc ccaagcaggg
      601 ccgcatgacc aacaagatga aaagcaccaa aggcgccctg accttcagcc cctacctgct
      661 gagccacgtg atgggctacg gcttctacca cttcggcacc taccccagcg gctacgagaa
      721 ccccttcctg cacgccatca acaacggcgg ctacaccaac acccgcatcg agaagtacga
      781 ggacggcggc gtgctgcacg tgagcttcag ctaccgctac gaggccggcc gcgtgatcgg
      841 cgacttcaag gtggtgggca ccggcttccc cgaggacagc gtgatcttca ccgacaagat
      901 catccgcagc aacgccaccg tggagcacct gcaccccatg ggcgataacg tgctggtggg
      961 cagcttcgcc cgcaccttca gcctgcgcga cggcggctac tacagcttcg tggtggacag
     1021 ccacatgcac ttcaagagcg ccatccaccc cagcatcctg cagaacgggg gccccatgtt
     1081 cgccttccgc cgcgtggagg agctgcacag caacaccgag ctgggcatcg tggagtacca
     1141 gcacgccttc aagaccccca tcgccttcgc cagatcccgc gctcagtcgt ccaattctgc
     1201 cgtggacggc accgccggac ccggctccac cggatctcgc cttaagggca gcggcgccac
     1261 caacttcagc ctgctgaagc aggccggcga cgtggaggag aaccccggcc ccatgaccga
     1321 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     1381 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     1441 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     1501 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     1561 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     1621 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     1681 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     1741 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     1801 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     1861 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatccatgcc
     1921 cacgctactg cgggtttata tagacggtcc tcacgggatg gggaaaacca ccaccacgca
     1981 actgctggtg gccctgggtt cgcgcgacga tatcgtctac gtacccgagc cgatgactta
     2041 ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc tacaccacac aacaccgcct
     2101 cgaccagggt gagatatcgg ccggggacgc ggcggtggta atgacaagcg cccagataac
     2161 aatgggcatg ccttatgccg tgaccgacgc cgttctggct cctcatatcg ggggggaggc
     2221 tgggagctca catgccccgc ccccggccct caccctcatc ttcgaccgcc atcccatcgc
     2281 cgccctcctg tgctacccgg ccgcgcgata ccttatgggc agcatgaccc cccaggccgt
     2341 gctggcgttc gtggccctca tcccgccgac cttgcccggc acaaacatcg tgttgggggc
     2401 ccttccggag gacagacaca tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct
     2461 tgacctggct atgctggccg cgattcgccg cgtttacggg ctgcttgcca atacggtgcg
     2521 gtatctgcag ggcggcgggt cgtggcggga ggattgggga cagctttcgg ggacggccgt
     2581 gccgccccag ggtgccgagc cccagagcaa cgcgggccca cgaccccata tcggggacac
     2641 gttatttacc ctgtttcggg cccccgagtt gctggccccc aacggcgacc tgtacaacgt
     2701 gtttgcctgg gccttggacg tcttggccaa acgcctccgt cccatgcacg tctttatcct
     2761 ggattacgac caatcgcccg ccggctgccg ggacgccctg ctgcaactta cctccgggat
     2821 ggtccagacc cacgtcacca cccccggctc cataccgacg atctgcgacc tggcgcgcac
     2881 gtttgcccgg gagatggggg aggctaactg actcgaggcc ggcaaggccg gatccagaca
     2941 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     3001 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     3061 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     3121 ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat ccggctgcct
     3181 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3241 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3301 tggcgggtgt cggggcgcag ccatgaggtc gactctagag gatcgatccc cgccccggac
     3361 gaactaaacc tgactacgac atctctgccc cttcttcgcg gggcagtgca tgtaatccct
     3421 tcagttggtt ggtacaactt gccaactggg ccctgttcca catgtgacac ggggggggac
     3481 caaacacaaa ggggttctct gactgtagtt gacatcctta taaatggatg tgcacatttg
     3541 ccaacactga gtggctttca tcctggagca gactttgcag tctgtggact gcaacacaac
     3601 attgccttta tgtgtaactc ttggctgaag ctcttacacc aatgctgggg gacatgtacc
     3661 tcccaggggc ccaggaagac tacgggaggc tacaccaacg tcaatcagag gggcctgtgt
     3721 agctaccgat aagcggaccc tcaagagggc attagcaata gtgtttataa ggcccccttg
     3781 ttaaccctaa acgggtagca tatgcttccc gggtagtagt atatactatc cagactaacc
     3841 ctaattcaat agcatatgtt acccaacggg aagcatatgc tatcgaatta gggttagtaa
     3901 aagggtccta aggaacagcg atatctccca ccccatgagc tgtcacggtt ttatttacat
     3961 ggggtcagga ttccacgagg gtagtgaacc attttagtca caagggcagt ggctgaagat
     4021 caaggagcgg gcagtgaact ctcctgaatc ttcgcctgct tcttcattct ccttcgttta
     4081 gctaatagaa taactgctga gttgtgaaca gtaaggtgta tgtgaggtgc tcgaaaacaa
     4141 ggtttcaggt gacgccccca gaataaaatt tggacggggg gttcagtggt ggcattgtgc
     4201 tatgacacca atataaccct cacaaacccc ttgggcaata aatactagtg taggaatgaa
     4261 acattctgaa tatctttaac aatagaaatc catggggtgg ggacaagccg taaagactgg
     4321 atgtccatct cacacgaatt tatggctatg ggcaacacat aatcctagtg caatatgata
     4381 ctggggttat taagatgtgt cccaggcagg gaccaagaca ggtgaaccat gttgttacac
     4441 tctatttgta acaaggggaa agagagtgga cgccgacagc agcggactcc actggttgtc
     4501 tctaacaccc ccgaaaatta aacggggctc cacgccaatg gggcccataa acaaagacaa
     4561 gtggccactc ttttttttga aattgtggag tgggggcacg cgtcagcccc cacacgccgc
     4621 cctgcggttt tggactgtaa aataagggtg taataacttg gctgattgta accccgctaa
     4681 ccactgcggt caaaccactt gcccacaaaa ccactaatgg caccccgggg aatacctgca
     4741 taagtaggtg ggcgggccaa gataggggcg cgattgctgc gatctggagg acaaattaca
     4801 cacacttgcg cctgagcgcc aagcacaggg ttgttggtcc tcatattcac gaggtcgctg
     4861 agagcacggt gggctaatgt tgccatgggt agcatatact acccaaatat ctggatagca
     4921 tatgctatcc taatctatat ctgggtagca takgctatcc taatctatat ctgggtagca
     4981 takgctatcc taatctatat ctgggtagya tatgctatcc taatytatat ctgggtagya
     5041 tatgctatcc taatytatat ctgggtagca takgctatcc taatctatat ctgggtagca
     5101 tatgctatcc taatctatat ctgggtagya tatgctatcc taatctgtat ccgggtagca
     5161 tatgctatcc tcatgcatat acagtcagca tatgataccc agtagtagag tgggagtgct
     5221 atcctttgca tatgccgcca cctcccaagg gggcgtgaat tttcgctgct tgtccttttc
     5281 ctgctggttg ctcccattct taggtgaatt taaggaggcc aggctaaagc cgtcgcatgt
     5341 ctgattgctc accaggtaaa tgtcgctaat gttttccaac gcgagaaggt gaattctaga
     5401 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5461 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5521 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5581 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5641 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5701 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5761 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5821 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5881 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5941 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6001 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6061 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     6121 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6181 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6241 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6301 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6361 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6421 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6481 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6541 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6601 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6661 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     6721 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6781 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6841 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6901 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6961 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7021 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7081 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7141 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7201 cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg
     7261 gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
     7321 tcgaactaga tgatccggct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     7381 tccccagcag gcagaagtat gcaaagcatg cgagtttact ccctatcagt gatagagaac
     7441 gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta ctccctatca
     7501 gtgatagaga acgtatgtc
//
