LOCUS       sequence-9549-ex        9675 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 9675)
  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 9675)
  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: pnlacZ-ACN
FEATURES             Location/Qualifiers
     source          1..9675
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..53
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        34..52
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             315..335
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3184..3578
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     protein_bind    3590..3623
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3650..3698
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(3705..4508)
                     /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(3816..3835)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4426..4445
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     polyA_signal    5247..5295
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     intron          5645..5710
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(6536..6556)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6580..6591)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    7266..7299
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(7363..7381)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7364..7381)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(7462..7481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7581..7603
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7641..7659)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7727..7831
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7832..7900,7901..8692)
                     /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(8050..8069)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8863..9451
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9352..9371
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9605..9622
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcattaatgc aggttaacct ggcttatcga aattaatacg actcactata gggagaccgg
       61 cagatccggc gcgcctgcag gccatggacc ctgttgtgct gcaaaggaga gactgggaga
      121 accctggagt gacccagctc aacagactgg ctgcccaccc tccctttgcc tcttggagga
      181 actctgagga agccaggaca gacaggccca gccagcagct caggtctctc aatggagagt
      241 ggaggtttgc ctggttccct gcccctgaag ctgtgcctga gtcttggctg gagtgtgacc
      301 tcccagaggc agttcccaag aagaagagga aagttgaggc tgacactgtt gtggtgccaa
      361 gcaactggca gatgcatggc tatgatgccc ccatctacac caatgtcacc taccccatca
      421 ctgtgaaccc cccttttgtg cccactgaga accccactgg ctgctacagc ctgaccttca
      481 atgttgatga gagctggctg caagaaggcc agaccaggat catctttgat ggagtcaact
      541 ctgccttcca cctctggtgc aatggcaggt gggttggcta tggccaagac agcaggctgc
      601 cctctgagtt tgacctctct gccttcctca gagctggaga gaacaggctg gctgtcatgg
      661 tgctcaggtg gtctgatggc agctacctgg aagaccaaga catgtggagg atgtctggca
      721 tcttcaggga tgtgagcctg ctgcacaagc ccaccaccca gatttctgac ttccatgttg
      781 ccaccaggtt caatgatgac ttcagcagag ctgtgctgga ggctgaggtg cagatgtgtg
      841 gagaactcag agactacctg agagtcacag tgagcctctg gcaaggtgag acccaggtgg
      901 cctctggcac agcccccttt ggaggagaga tcattgatga gagaggaggc tatgctgaca
      961 gagtcaccct gaggctcaat gtggagaacc ccaagctgtg gtctgctgag atccccaacc
     1021 tctacagggc tgttgtggag ctgcacactg ctgatggcac cctgattgaa gctgaagcct
     1081 gtgatgttgg attcagagaa gtcaggattg agaatggcct gctgctgctc aatggcaagc
     1141 ctctgctcat caggggagtc aacaggcatg agcaccaccc tctgcatgga caagtgatgg
     1201 atgaacagac aatggtgcaa gatatcctgc taatgaagca gaacaacttc aatgctgtca
     1261 ggtgctctca ctaccccaac caccctctct ggtacaccct gtgtgacagg tatggcctgt
     1321 atgttgttga tgaagccaac attgagacac atggcatggt gcccatgaac aggctcacag
     1381 atgaccccag gtggctgcct gccatgtctg agagagtgac caggatggtg cagagagaca
     1441 ggaaccaccc ctctgtgatc atctggtctc tgggcaatga gtctggacat ggagccaacc
     1501 atgatgctct ctacaggtgg atcaagtctg ttgaccccag cagacctgtg cagtatgaag
     1561 gaggtggagc agacaccaca gccacagaca tcatctgccc catgtatgcc agggttgatg
     1621 aggaccagcc cttccctgct gtgcccaagt ggagcatcaa gaagtggctc tctctgcctg
     1681 gagagaccag acctctgatc ctgtgtgaat atgcacatgc aatgggcaac tctctgggag
     1741 gctttgccaa gtactggcaa gccttcagac agtaccccag gctgcaagga ggatttgtgt
     1801 gggactgggt ggaccaatct ctcatcaagt atgatgagaa tggcaacccc tggtctgcct
     1861 atggaggaga ctttggtgac acccccaatg acaggcagtt ctgcatgaat ggcctggtct
     1921 ttgcagacag gacccctcac cctgccctca cagaggccaa gcaccagcaa cagttcttcc
     1981 agttcaggct gtctggacag accattgagg tgacatctga gtacctcttc aggcactctg
     2041 acaatgagct cctgcactgg atggtggccc tggatggcaa gcctctggct tctggtgagg
     2101 tgcctctgga tgtggcccct caaggaaagc agctgattga actgcctgag ctgcctcagc
     2161 cagagtctgc tggacaactg tggctaacag tgagggtggt tcagcccaat gcaacagctt
     2221 ggtctgaggc aggccacatc tctgcatggc agcagtggag gctggctgag aacctctctg
     2281 tgaccctgcc tgctgcctct catgccatcc ctcacctgac aacatctgaa atggacttct
     2341 gcattgagct gggcaacaag agatggcagt tcaacaggca gtctggcttc ctgtctcaga
     2401 tgtggattgg agacaagaag cagctcctca cccctctcag ggaccaattc accagggctc
     2461 ctctggacaa tgacattgga gtgtctgagg ccaccaggat tgacccaaat gcttgggtgg
     2521 agaggtggaa ggctgctgga cactaccagg ctgaggctgc cctgctccag tgcacagcag
     2581 acaccctggc tgatgctgtt ctgatcacca cagcccatgc ttggcagcac caaggcaaga
     2641 ccctgttcat cagcagaaag acctacagga ttgatggctc tggacagatg gcaatcacag
     2701 tggatgtgga ggttgcctct gacacacctc accctgcaag gattggcctg aactgtcaac
     2761 tggcacaggt ggctgagagg gtgaactggc tgggcttagg ccctcaggag aactaccctg
     2821 acaggctgac agctgcctgc tttgacaggt gggacctgcc tctgtctgac atgtacaccc
     2881 cttatgtgtt cccttctgag aatggcctga ggtgtggcac cagggagctg aactatggtc
     2941 ctcaccagtg gaggggagac ttccagttca acatctccag gtactctcag caacagctca
     3001 tggaaacctc tcacaggcac ctgctccatg cagaggaggg aacctggctg aacattgatg
     3061 gcttccacat gggcattgga ggagatgact cttggtctcc ttctgtgtct gctgagttcc
     3121 agttatctgc tggcaggtac cactatcagc tggtgtggtg ccagaagtaa acctaatcta
     3181 gaagctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc taagtccaac
     3241 tactaaactg ggggatatta tgaagggcct tgagcatctg gattctgcct aataaaaaac
     3301 atttattttc attgcaatga tgtatttaaa ttatttctga atattttact aaaaagggaa
     3361 tgtgggaggt cagtgcattt aaaacataaa gaaatgaaga gctagttcaa accttgggaa
     3421 aatacactat atcttaaact ccatgaaaga aggtgaggct gcaaacagct aatgcacatt
     3481 ggcaacagcc cctgatgcct atgccttatt catccctcag aaaaggattc aagtagaggc
     3541 ttgatttgga ggttaaagtt ttgctatgct gtattttaat taagaattca taacttcgta
     3601 taatgtatgc tatacgaagt tatggatctg tcgatcgacg gatcgatccg aacaaacgac
     3661 ccaacacccg tgcgttttat tctgtctttt tattgccgat cccctcagaa gaactcgtca
     3721 agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg
     3781 aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg
     3841 tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca
     3901 ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg
     3961 tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct
     4021 tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg
     4081 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc
     4141 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc
     4201 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc
     4261 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc
     4321 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct
     4381 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg
     4441 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatggcc
     4501 gatcccatat tggctgcacg gatcctgaac ggcagaggtt acggcagttt gtctctcccc
     4561 cttccgggag ccaccttctt ctccaaccgt cccggtcgcg ctctcggcgc ttctgaggag
     4621 agaactggct gagtgacgcc ctttatagat tcgcccttgt gtcccgcccc ttcctttccc
     4681 gccctccctt gcgctacggg gccgcccgca ccggcctaca cggagcgcgc gcggcggagt
     4741 tgttgacgct agggctccgg ctccctggtt gggtgttctt tctgacgcga caggaggagg
     4801 agaatgtcct ggtcctgtcg tcctcctttc gggtttcccg tgcactcaaa ccgaggactt
     4861 acagaacgga ggataaagtt aggccatttt tactcagctt cggagttcag gctcattttt
     4921 cagctaaagt ctctcattag tatcccccca cacacatcgg gaaaatggtt tgtcctacgc
     4981 atcggtaatg aaggcggggc ccttcgggtc ctccggagcg ggttccgggg gtggggggaa
     5041 ggagggaggg acgggacggg cctcgttcat gaatattcag ttcaccgctg aatatgcata
     5101 aggcaggcaa gatggcgcgt ccaatcaatt ggaagtagcc gttattagtg gagaggcccc
     5161 aggacgttgg ggcaccgcct gtgctctagt agctttacgg agccctggcg ctcgatgttc
     5221 aagcccaagc tttcgcgagc tcgaccgaac aaacgaccca acacccgtgc gttttattct
     5281 gtctttttat tgccgctcag ctttacagtg acaatgacgg ctggcgactg aatattagtg
     5341 cttacagaca gcactacata ttttccgtcg atgttgaaat cctttctcat atgtcaccat
     5401 aaatatcaaa taattatagc aatcatttac gcgttaatgg ctaatcgcca tcttccagca
     5461 ggcgcaccat tgcccctgtt tcactatcca ggttacggat atagttcatg acaatattta
     5521 cattggtcca gccaccagct tgcatgatct ccggtattga aactccagcg cgggccatat
     5581 ctcgcgcggc tccgacacgg gcactgtgtc cagaccaggc caggtatctc tgaccagagt
     5641 catcctaaaa tacacaaaca attagaatca gtagtttaac acattataca cttaaaaatt
     5701 ttatatttac cttagcgccg taaatcaatc gatgagttgc ttcaaaaatc ccttccaggg
     5761 cgcgagttga tagctggctg gtggcagatg gcgcggcaac accatttttt ctgacccggc
     5821 aaaacaggta gttattcgga tcatcagcta caccagagac ggaaatccat cgctcgacca
     5881 gtttagttac ccccaggcta agtgccttct ctacacctgc ggtgctaacc agcgttttcg
     5941 ttctgccaat atggattaac attctcccac cgtcagtacg tgagatatct ttaaccctga
     6001 tcctggcaat ttcggctata cgtaacaggg tgttataagc aatccccaga aatgccagat
     6061 tacgtatatc ctggcagcga tcgctatttt ccatgagtga acgaacctgg tcgaaatcag
     6121 tgcgttcgaa cgctagagcc tgttttgcac gttcaccggc atcaacgttt tcttttcgga
     6181 tccgccgcat aaccagtgaa acagcattgc tgtcacttgg tcgtggcagc ccggaccgac
     6241 gatgaagcat gtttagctgg cccaaatgtt gctggatagt ttttactgcc agaccgcgcg
     6301 cctgaagata tagaagataa tcgcgaacat cttcaggttc tgcgggaaac catttccggt
     6361 tattcaactt gcaccatgcc gcccacgacc ggcaaacgga cagaagcatt ttccaggtat
     6421 gctcagaaaa cgcctggcga tccctgaaca tgtccatcag gttcttgcga acctcatcac
     6481 tcgttgcatc gaccggtaat gcaggcaaat tttggtgtac ggtcagtaaa ttggacacct
     6541 tcctcttctt cttgggcatg gccgcaggaa agcagagccc tgaagctccc atcaccggcc
     6601 aataagagcc aagcctgcag tgtgacctca tagagcaatg tgccagccag cctgacccca
     6661 agggccctca ggcttgggca cactgtctct aggaccctga gagaaagaca tacccatttc
     6721 tgcttagggc cctgaggatg agcccagggg tggcttggca ctgaagcaaa ggacactggg
     6781 gctcagctgg cagcaaagtg accaggatgc tgaggctttg acccagaagc cagaggccag
     6841 aggccaggac ttctcttggt cccagtccac cctcactcag agctttacca atgccctctg
     6901 gatagttgtc gggtaacggt ggacgccact gattctctgg ccagcctagg acttcgccat
     6961 tccgctgatt ctgctcttcc agccactggc tgaccggttg gaagtactcc agcagtgcct
     7021 tggcatccag ggcatctgag cctaccaggt ccttcagtac ctcctgccag ggcctggagc
     7081 agccagcctg caacacctgc ctgccaagca gagtgaccac tgtgggcaca ggggacacag
     7141 ggtggggccc acaacagcac cattgtccac ttgtccctca ctagtaaaag aactctaggg
     7201 ttgcgggggg tgggggaggt ctctgtgagg ctggtaaggg atatttgcct ggcccatgga
     7261 gatccataac ttcgtataat gtatgctata cgaagttata agctttcgcg agctcgagat
     7321 cccagtcagt cagtctcgag cgatcgcggc gcgccgtcga gttctatagt gtcacctaaa
     7381 tcgtatgtgt atgatacata aggttatgta ttaattgtag ccgcgttcta acgacaatat
     7441 gtccatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     7501 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     7561 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     7621 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     7681 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     7741 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     7801 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     7861 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     7921 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     7981 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     8041 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     8101 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     8161 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     8221 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     8281 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     8341 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     8401 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     8461 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     8521 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     8581 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     8641 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     8701 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     8761 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     8821 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     8881 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     8941 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     9001 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     9061 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     9121 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     9181 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     9241 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     9301 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     9361 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     9421 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     9481 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     9541 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     9601 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     9661 cgcgcgttgg ccgat
//
