LOCUS       sequence-9548-ex        7904 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 7904)
  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 7904)
  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: pECFPpA-ACN
FEATURES             Location/Qualifiers
     source          1..7904
                     /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="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     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    3456..3504
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     intron          3854..3919
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(4745..4765)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4789..4800)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    5475..5508
                     /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(5558..5577)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5559..5577)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5664..5681)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5835..6423)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5915..5934)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6594..7385,7386..7454))
                     /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     7217..7236
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7455..7559)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7627..7645
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7683..7705)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7805..7824
                     /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 cctgacctgg 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 caactacatc agccacaacg
     1261 tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag 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 gaatgtcctg gtcctgtcgt cctcctttcg ggtttcccgt gcactcaaac
     3061 cgaggactta cagaacggag gataaagtta ggccattttt actcagcttc ggagttcagg
     3121 ctcatttttc 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 agcccaagct ttcgcgagct cgaccgaaca aacgacccaa cacccgtgcg
     3481 ttttattctg tctttttatt gccgctcagc tttacagtga caatgacggc tggcgactga
     3541 atattagtgc ttacagacag cactacatat tttccgtcga tgttgaaatc ctttctcata
     3601 tgtcaccata aatatcaaat aattatagca atcatttacg cgttaatggc taatcgccat
     3661 cttccagcag gcgcaccatt gcccctgttt cactatccag gttacggata tagttcatga
     3721 caatatttac attggtccag ccaccagctt gcatgatctc cggtattgaa actccagcgc
     3781 gggccatatc tcgcgcggct ccgacacggg cactgtgtcc agaccaggcc aggtatctct
     3841 gaccagagtc atcctaaaat acacaaacaa ttagaatcag tagtttaaca cattatacac
     3901 ttaaaaattt tatatttacc ttagcgccgt aaatcaatcg atgagttgct tcaaaaatcc
     3961 cttccagggc gcgagttgat agctggctgg tggcagatgg cgcggcaaca ccattttttc
     4021 tgacccggca aaacaggtag ttattcggat catcagctac accagagacg gaaatccatc
     4081 gctcgaccag tttagttacc cccaggctaa gtgccttctc tacacctgcg gtgctaacca
     4141 gcgttttcgt tctgccaata tggattaaca ttctcccacc gtcagtacgt gagatatctt
     4201 taaccctgat cctggcaatt tcggctatac gtaacagggt gttataagca atccccagaa
     4261 atgccagatt acgtatatcc tggcagcgat cgctattttc catgagtgaa cgaacctggt
     4321 cgaaatcagt gcgttcgaac gctagagcct gttttgcacg ttcaccggca tcaacgtttt
     4381 cttttcggat ccgccgcata accagtgaaa cagcattgct gtcacttggt cgtggcagcc
     4441 cggaccgacg atgaagcatg tttagctggc ccaaatgttg ctggatagtt tttactgcca
     4501 gaccgcgcgc ctgaagatat agaagataat cgcgaacatc ttcaggttct gcgggaaacc
     4561 atttccggtt attcaacttg caccatgccg cccacgaccg gcaaacggac agaagcattt
     4621 tccaggtatg ctcagaaaac gcctggcgat ccctgaacat gtccatcagg ttcttgcgaa
     4681 cctcatcact cgttgcatcg accggtaatg caggcaaatt ttggtgtacg gtcagtaaat
     4741 tggacacctt cctcttcttc ttgggcatgg ccgcaggaaa gcagagccct gaagctccca
     4801 tcaccggcca ataagagcca agcctgcagt gtgacctcat agagcaatgt gccagccagc
     4861 ctgaccccaa gggccctcag gcttgggcac actgtctcta ggaccctgag agaaagacat
     4921 acccatttct gcttagggcc ctgaggatga gcccaggggt ggcttggcac tgaagcaaag
     4981 gacactgggg ctcagctggc agcaaagtga ccaggatgct gaggctttga cccagaagcc
     5041 agaggccaga ggccaggact tctcttggtc ccagtccacc ctcactcaga gctttaccaa
     5101 tgccctctgg atagttgtcg ggtaacggtg gacgccactg attctctggc cagcctagga
     5161 cttcgccatt ccgctgattc tgctcttcca gccactggct gaccggttgg aagtactcca
     5221 gcagtgcctt ggcatccagg gcatctgagc ctaccaggtc cttcagtacc tcctgccagg
     5281 gcctggagca gccagcctgc aacacctgcc tgccaagcag agtgaccact gtgggcacag
     5341 gggacacagg gtggggccca caacagcacc attgtccact tgtccctcac tagtaaaaga
     5401 actctagggt tgcggggggt gggggaggtc tctgtgaggc tggtaaggga tatttgcctg
     5461 gcccatggag atccataact tcgtataatg tatgctatac gaagttataa gctttcgcga
     5521 gctcgagatc ctgcaggcgc gccggatctg ccggtctccc tatagtgagt cgtattaatt
     5581 tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5641 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5701 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5761 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5821 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5881 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5941 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     6001 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6061 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6121 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6181 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6241 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6301 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6361 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6421 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6481 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6541 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6601 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6661 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6721 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6781 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6841 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6901 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6961 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     7021 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7081 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7141 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7201 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7261 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7321 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7381 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7441 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7501 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7561 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     7621 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     7681 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     7741 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     7801 tatgcggcat cagagcagat tgtactgaga gtgcaccata tggacatatt gtcgttagaa
     7861 cgcggctaca attaatacat aaccttatgt atcatacaca tacg
//
