LOCUS       sequence-311612-        7903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7903)
  AUTHORS   Wu S, Ying G, Wu Q, Capecchi MR
  TITLE     A protocol for constructing gene targeting vectors: generating 
            knockout mice for the cadherin family and beyond.
  JOURNAL   Nat Protoc. 2008  . 3(6):1056-76.
  PUBMED    18546598
REFERENCE   2  (bases 1 to 7903)
  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: "Nat Protoc.
            2008  . 3(6):1056-76."
COMMENT     Sequence Label: pEGFPpA-ACN
FEATURES             Location/Qualifiers
     source          1..7903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             44..64
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             62..778
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /label=hrGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGNQ
                     LVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYED
                     GGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVGQ
                     VILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAIA
                     QLTSLGKPLGSLHEWV"
     CDS             join(813..815,816..1529)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(813..815,816..1526)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1116..1135)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1463..1484
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1652..1773
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1689..1708)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1743..1762
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1799..1832
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1859..1907
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(1914..2717)
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2025..2044)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2635..2654
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     polyA_signal    3455..3503
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     intron          3853..3918
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(4744..4764)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4788..4799)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    5474..5507
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5557..5576)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5558..5576)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5663..5680)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5834..6422)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5914..5933)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6593..7384,7385..7453))
                     /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     7216..7235
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7454..7558)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7626..7644
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7682..7704)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7804..7823
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa ctcgacggcg cgccgcgatt aaccccaaga agaagaggaa
       61 ggtgagcaag cagatcctga agaacaccgg cctgcaggag atcatgagct tcaaggtgaa
      121 cctggagggc gtggtgaaca accacgtgtt caccatggag ggctgcggca agggcaacat
      181 cctgttcggc aaccagctgg tgcagatccg cgtgaccaag ggcgcccccc tgcccttcgc
      241 cttcgacatc ctgagccccg ccttccagta cggcaaccgc accttcacca agtaccccga
      301 ggacatcagc gacttcttca tccagagctt ccccgccggc ttcgtgtacg agcgcaccct
      361 gcgctacgag gacggcggcc tggtggagat ccgcagcgac atcaacctga tcgaggagat
      421 gttcgtgtac cgcgtggagt acaagggccg caacttcccc aacgacggcc ccgtgatgaa
      481 gaagaccatc accggcctgc agcccagctt cgaggtggtg tacatgaacg acggcgtgct
      541 ggtgggccag gtgatcctgg tgtaccgcct gaacagcggc aagttctaca gctgccacat
      601 gcgcaccctg atgaagagca agggcgtggt gaaggacttc cccgagtacc acttcatcca
      661 gcaccgcctg gagaagacct acgtggagga cggcggcttc gtggagcagc acgagaccgc
      721 catcgcccag ctgaccagcc tgggcaagcc cctgggcagc ctgcacgagt gggtgtaata
      781 ggaattcgcc cttgttaatt aagcggcgcg ccgtgagcaa gggcgaggag ctgttcaccg
      841 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      901 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      961 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1021 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1081 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1141 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1201 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1261 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1321 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1381 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1441 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1501 ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagatca taatcagcca
     1561 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     1621 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     1681 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     1741 ttgtggtttg tccaaactca tcaatgtatc ttaaggatcc gccgataagg gcgaattcat
     1801 aacttcgtat aatgtatgct atacgaagtt atggatctgt cgatcgacgg atcgatccga
     1861 acaaacgacc caacacccgt gcgttttatt ctgtcttttt attgccgatc ccctcagaag
     1921 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
     1981 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
     2041 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
     2101 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga
     2161 tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
     2221 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct
     2281 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
     2341 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
     2401 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
     2461 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
     2521 tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
     2581 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
     2641 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
     2701 tcaatggccg atcccatatt ggctgcacgg atcctgaacg gcagaggtta cggcagtttg
     2761 tctctccccc ttccgggagc caccttcttc tccaaccgtc ccggtcgcgc tctcggcgct
     2821 tctgaggaga gaactggctg agtgacgccc tttatagatt cgcccttgtg tcccgcccct
     2881 tcctttcccg ccctcccttg cgctacgggg ccgcccgcac cggcctacac ggagcgcgcg
     2941 cggcggagtt gttgacgcta gggctccggc tccctggttg ggtgttcttt ctgacgcgac
     3001 aggaggagga gaatgttcct ggtcctgtcg tcctcctttc gggtttcccg tgcactcaaa
     3061 ccgaggactt acagaacgga ggataaagtt aggccatttt tactcagctt cggagttcag
     3121 gctcattttc agctaaagtc tctcattagt atccccccac acacatcggg aaaatggttt
     3181 gtcctacgca tcggtaatga aggcggggcc cttcgggtcc tccggagcgg gttccggggg
     3241 tggggggaag gagggaggga cgggacgggc ctcgttcatg aatattcagt tcaccgctga
     3301 atatgcataa ggcaggcaag atggcgcgtc caatcaattg gaagtagccg ttattagtgg
     3361 agaggcccca ggacgttggg gcaccgcctg tgctctagta gctttacgga gccctggcgc
     3421 tcgatgttca gcccaagctt tcgcgagctc gaccgaacaa acgacccaac acccgtgcgt
     3481 tttattctgt ctttttattg ccgctcagct ttacagtgac aatgacggct ggcgactgaa
     3541 tattagtgct tacagacagc actacatatt ttccgtcgat gttgaaatcc tttctcatat
     3601 gtcaccataa atatcaaata attatagcaa tcatttacgc gttaatggct aatcgccatc
     3661 ttccagcagg cgcaccattg cccctgtttc actatccagg ttacggatat agttcatgac
     3721 aatatttaca ttggtccagc caccagcttg catgatctcc ggtattgaaa ctccagcgcg
     3781 ggccatatct cgcgcggctc cgacacgggc actgtgtcca gaccaggcca ggtatctctg
     3841 accagagtca tcctaaaata cacaaacaat tagaatcagt agtttaacac attatacact
     3901 taaaaatttt atatttacct tagcgccgta aatcaatcga tgagttgctt caaaaatccc
     3961 ttccagggcg cgagttgata gctggctggt ggcagatggc gcggcaacac cattttttct
     4021 gacccggcaa aacaggtagt tattcggatc atcagctaca ccagagacgg aaatccatcg
     4081 ctcgaccagt ttagttaccc ccaggctaag tgccttctct acacctgcgg tgctaaccag
     4141 cgttttcgtt ctgccaatat ggattaacat tctcccaccg tcagtacgtg agatatcttt
     4201 aaccctgatc ctggcaattt cggctatacg taacagggtg ttataagcaa tccccagaaa
     4261 tgccagatta cgtatatcct ggcagcgatc gctattttcc atgagtgaac gaacctggtc
     4321 gaaatcagtg cgttcgaacg ctagagcctg ttttgcacgt tcaccggcat caacgttttc
     4381 ttttcggatc cgccgcataa ccagtgaaac agcattgctg tcacttggtc gtggcagccc
     4441 ggaccgacga tgaagcatgt ttagctggcc caaatgttgc tggatagttt ttactgccag
     4501 accgcgcgcc tgaagatata gaagataatc gcgaacatct tcaggttctg cgggaaacca
     4561 tttccggtta ttcaacttgc accatgccgc ccacgaccgg caaacggaca gaagcatttt
     4621 ccaggtatgc tcagaaaacg cctggcgatc cctgaacatg tccatcaggt tcttgcgaac
     4681 ctcatcactc gttgcatcga ccggtaatgc aggcaaattt tggtgtacgg tcagtaaatt
     4741 ggacaccttc ctcttcttct tgggcatggc cgcaggaaag cagagccctg aagctcccat
     4801 caccggccaa taagagccaa gcctgcagtg tgacctcata gagcaatgtg ccagccagcc
     4861 tgaccccaag ggccctcagg cttgggcaca ctgtctctag gaccctgaga gaaagacata
     4921 cccatttctg cttagggccc tgaggatgag cccaggggtg gcttggcact gaagcaaagg
     4981 acactggggc tcagctggca gcaaagtgac caggatgctg aggctttgac ccagaagcca
     5041 gaggccagag gccaggactt ctcttggtcc cagtccaccc tcactcagag ctttaccaat
     5101 gccctctgga tagttgtcgg gtaacggtgg acgccactga ttctctggcc agcctaggac
     5161 ttcgccattc cgctgattct gctcttccag ccactggctg accggttgga agtactccag
     5221 cagtgccttg gcatccaggg catctgagcc taccaggtcc ttcagtacct cctgccaggg
     5281 cctggagcag ccagcctgca acacctgcct gccaagcaga gtgaccactg tgggcacagg
     5341 ggacacaggg tggggcccac aacagcacca ttgtccactt gtccctcact agtaaaagaa
     5401 ctctagggtt gcggggggtg ggggaggtct ctgtgaggct ggtaagggat atttgcctgg
     5461 cccatggaga tccataactt cgtataatgt atgctatacg aagttataag ctttcgcgag
     5521 ctcgagatcc tgcaggcgcg ccggatctgc cggtctccct atagtgagtc gtattaattt
     5581 cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5641 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5701 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5761 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5821 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5881 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5941 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6001 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6061 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6121 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6181 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6241 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6301 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6361 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6421 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6481 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6541 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6601 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6661 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6721 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6781 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6841 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6901 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6961 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7021 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7081 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7141 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7201 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7261 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7321 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7381 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7441 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7501 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7561 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     7621 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     7681 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     7741 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     7801 atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac
     7861 gcggctacaa ttaatacata accttatgta tcatacacat acg
//
