LOCUS       sequence-9547-ex        8196 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 8196)
  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 8196)
  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: pAP-ACN
FEATURES             Location/Qualifiers
     source          1..8196
                     /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             87..1586
                     /codon_start=1
                     /product="secreted alkaline phosphatase from human 
                     placenta"
                     /label=SEAP
                     /note="The natural enzyme has a C-terminal transmembrane 
                     sequence that is replaced by a GPI anchor."
                     /translation="LLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPA
                     QTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKH
                     VPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVT
                     TTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKY
                     MFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHL
                     MGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRA
                     YRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKAR
                     DRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFAR
                     GPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTD"
     polyA_signal    1705..2099
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     protein_bind    2111..2144
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2171..2219
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(2226..3029)
                     /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(2337..2356)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2947..2966
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     polyA_signal    3768..3816
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     intron          4166..4231
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5057..5077)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5101..5112)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    5787..5820
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(5884..5902)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5885..5902)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5983..6002)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6102..6124
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6162..6180)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6248..6352
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6353..6421,6422..7213)
                     /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(6571..6590)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7384..7972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7873..7892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8126..8143
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcattaatgc aggttaacct ggcttatcga aattaatacg actcactata gggagaccgg
       61 cagatccggc gcgcctgcag gatctcctgc tgctgctgct gctgctgggc ctgaggctac
      121 agctctccct gggcatcatc ccagttgagg aggagaaccc ggacttctgg aaccgcgagg
      181 cagccgaggc cctgggtgcc gccaagaagc tgcagcctgc acagacagcc gccaagaacc
      241 tcatcatctt cctgggcgat gggatggggg tgtctacggt gacagctgcc aggatcttaa
      301 aagggcagaa gaaggacaaa ctggggcctg agatacccct ggccatggac cgcttcccat
      361 atgtggctct gtccaagaca tacaatgtag acaaacatgt gccagacagt ggagccacag
      421 ccacggccta cctgtgcggg gtcaagggca acttccagac cattggcttg agtgcagccg
      481 cccgctttaa ccagtgcaac acgacacgcg gcaacgaggt catctccgtg atgaatcggg
      541 ccaagaaagc agggaagtca gtgggagtgg taaccaccac acgagtgcag cacgcctcgc
      601 cagccggcac ctacgcccac acggtgaacc gcaactggta ctcggacgcc gacgtgcctg
      661 cctcggcccg ccaggagggg tgccaggaca tcgctacgca gctcatctcc aacatggaca
      721 ttgacgtgat cctaggtgga ggccgaaagt acatgtttcg catgggaacc ccagaccctg
      781 agtacccaga tgactacagc caaggtggga ccaggctgga cgggaagaat ctggtgcagg
      841 aatggctggc gaagcgccag ggtgcccggt atgtgtggaa ccgcactgag ctcatgcagg
      901 cttccctgga cccgtctgtg acccatctca tgggtctctt tgagcctgga gacatgaaat
      961 acgagatcca ccgagactcc acactggacc cctccctgat ggagatgaca gaggctgccc
     1021 tgcgcctgct gagcaggaac ccccgcggct tcttcctctt cgtggagggt ggtcgcatcg
     1081 accatggtca tcatgaaagc agggcttacc gggcactgac tgagacgatc atgttcgacg
     1141 acgccattga gagggcgggc cagctcacca gcgaggagga cacgctgagc ctcgtcactg
     1201 ccgaccactc ccacgtcttc tccttcggag gctaccccct gcgagggagc tccatcttcg
     1261 ggctggcccc tggcaaggcc cgggacagga aggcctacac ggtcctccta tacggaaacg
     1321 gtccaggcta tgtgctcaag gacggcgccc ggccggatgt taccgagagc gagagcggga
     1381 gccccgagta tcggcagcag tcagcagtgc ccctggacga agagacccac gcaggcgagg
     1441 acgtggcggt gttcgcgcgc ggcccgcagg cgcacctggt tcacggcgtg caggagcaga
     1501 ccttcatagc gcacgtcatg gccttcgccg cctgcctgga gccctacacc gcctgcgacc
     1561 tggcgccccc cgccggcacc accgacgccg cgcacccggg gcggtccgtg gtccccgcgt
     1621 tgcttcctct gctggccggg accctgctgc tgctggagac ggccactgct ccctgagatc
     1681 gaattaattc gatagcttct agaagctcgc tttcttgctg tccaatttct attaaaggtt
     1741 cctttgttcc ctaagtccaa ctactaaact gggggatatt atgaagggcc ttgagcatct
     1801 ggattctgcc taataaaaaa catttatttt cattgcaatg atgtatttaa attatttctg
     1861 aatattttac taaaaaggga atgtgggagg tcagtgcatt taaaacataa agaaatgaag
     1921 agctagttca aaccttggga aaatacacta tatcttaaac tccatgaaag aaggtgaggc
     1981 tgcaaacagc taatgcacat tggcaacagc ccctgatgcc tatgccttat tcatccctca
     2041 gaaaaggatt caagtagagg cttgatttgg aggttaaagt tttgctatgc tgtattttaa
     2101 ttaagaattc ataacttcgt ataatgtatg ctatacgaag ttatggatct gtcgatcgac
     2161 ggatcgatcc gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccga
     2221 tcccctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg
     2281 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata
     2341 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg
     2401 atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg
     2461 gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct
     2521 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc
     2581 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga
     2641 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca
     2701 aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc
     2761 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat
     2821 agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa
     2881 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc
     2941 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc
     3001 aatccatctt gttcaatggc cgatcccata ttggctgcac ggatcctgaa cggcagaggt
     3061 tacggcagtt tgtctctccc ccttccggga gccaccttct tctccaaccg tcccggtcgc
     3121 gctctcggcg cttctgagga gagaactggc tgagtgacgc cctttataga ttcgcccttg
     3181 tgtcccgccc cttcctttcc cgccctccct tgcgctacgg ggccgcccgc accggcctac
     3241 acggagcgcg cgcggcggag ttgttgacgc tagggctccg gctccctggt tgggtgttct
     3301 ttctgacgcg acaggaggag gagaatgtcc tggtcctgtc gtcctccttt cgggtttccc
     3361 gtgcactcaa accgaggact tacagaacgg aggataaagt taggccattt ttactcagct
     3421 tcggagttca ggctcatttt tcagctaaag tctctcatta gtatcccccc acacacatcg
     3481 ggaaaatggt ttgtcctacg catcggtaat gaaggcgggg cccttcgggt cctccggagc
     3541 gggttccggg ggtgggggga aggagggagg gacgggacgg gcctcgttca tgaatattca
     3601 gttcaccgct gaatatgcat aaggcaggca agatggcgcg tccaatcaat tggaagtagc
     3661 cgttattagt ggagaggccc caggacgttg gggcaccgcc tgtgctctag tagctttacg
     3721 gagccctggc gctcgatgtt caagcccaag ctttcgcgag ctcgaccgaa caaacgaccc
     3781 aacacccgtg cgttttattc tgtcttttta ttgccgctca gctttacagt gacaatgacg
     3841 gctggcgact gaatattagt gcttacagac agcactacat attttccgtc gatgttgaaa
     3901 tcctttctca tatgtcacca taaatatcaa ataattatag caatcattta cgcgttaatg
     3961 gctaatcgcc atcttccagc aggcgcacca ttgcccctgt ttcactatcc aggttacgga
     4021 tatagttcat gacaatattt acattggtcc agccaccagc ttgcatgatc tccggtattg
     4081 aaactccagc gcgggccata tctcgcgcgg ctccgacacg ggcactgtgt ccagaccagg
     4141 ccaggtatct ctgaccagag tcatcctaaa atacacaaac aattagaatc agtagtttaa
     4201 cacattatac acttaaaaat tttatattta ccttagcgcc gtaaatcaat cgatgagttg
     4261 cttcaaaaat cccttccagg gcgcgagttg atagctggct ggtggcagat ggcgcggcaa
     4321 caccattttt tctgacccgg caaaacaggt agttattcgg atcatcagct acaccagaga
     4381 cggaaatcca tcgctcgacc agtttagtta cccccaggct aagtgccttc tctacacctg
     4441 cggtgctaac cagcgttttc gttctgccaa tatggattaa cattctccca ccgtcagtac
     4501 gtgagatatc tttaaccctg atcctggcaa tttcggctat acgtaacagg gtgttataag
     4561 caatccccag aaatgccaga ttacgtatat cctggcagcg atcgctattt tccatgagtg
     4621 aacgaacctg gtcgaaatca gtgcgttcga acgctagagc ctgttttgca cgttcaccgg
     4681 catcaacgtt ttcttttcgg atccgccgca taaccagtga aacagcattg ctgtcacttg
     4741 gtcgtggcag cccggaccga cgatgaagca tgtttagctg gcccaaatgt tgctggatag
     4801 tttttactgc cagaccgcgc gcctgaagat atagaagata atcgcgaaca tcttcaggtt
     4861 ctgcgggaaa ccatttccgg ttattcaact tgcaccatgc cgcccacgac cggcaaacgg
     4921 acagaagcat tttccaggta tgctcagaaa acgcctggcg atccctgaac atgtccatca
     4981 ggttcttgcg aacctcatca ctcgttgcat cgaccggtaa tgcaggcaaa ttttggtgta
     5041 cggtcagtaa attggacacc ttcctcttct tcttgggcat ggccgcagga aagcagagcc
     5101 ctgaagctcc catcaccggc caataagagc caagcctgca gtgtgacctc atagagcaat
     5161 gtgccagcca gcctgacccc aagggccctc aggcttgggc acactgtctc taggaccctg
     5221 agagaaagac atacccattt ctgcttaggg ccctgaggat gagcccaggg gtggcttggc
     5281 actgaagcaa aggacactgg ggctcagctg gcagcaaagt gaccaggatg ctgaggcttt
     5341 gacccagaag ccagaggcca gaggccagga cttctcttgg tcccagtcca ccctcactca
     5401 gagctttacc aatgccctct ggatagttgt cgggtaacgg tggacgccac tgattctctg
     5461 gccagcctag gacttcgcca ttccgctgat tctgctcttc cagccactgg ctgaccggtt
     5521 ggaagtactc cagcagtgcc ttggcatcca gggcatctga gcctaccagg tccttcagta
     5581 cctcctgcca gggcctggag cagccagcct gcaacacctg cctgccaagc agagtgacca
     5641 ctgtgggcac aggggacaca gggtggggcc cacaacagca ccattgtcca cttgtccctc
     5701 actagtaaaa gaactctagg gttgcggggg gtgggggagg tctctgtgag gctggtaagg
     5761 gatatttgcc tggcccatgg agatccataa cttcgtataa tgtatgctat acgaagttat
     5821 aagctttcgc gagctcgaga tcccagtcag tcagtctcga gcgatcgcgg cgcgccgtcg
     5881 agttctatag tgtcacctaa atcgtatgtg tatgatacat aaggttatgt attaattgta
     5941 gccgcgttct aacgacaata tgtccatatg gtgcactctc agtacaatct gctctgatgc
     6001 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     6061 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     6121 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     6181 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     6241 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     6301 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     6361 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     6421 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     6481 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     6541 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6601 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6661 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6721 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6781 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6841 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6901 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6961 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     7021 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     7081 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     7141 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     7201 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     7261 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     7321 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7381 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7441 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     7501 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     7561 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7621 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7681 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7741 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7801 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7861 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7921 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7981 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     8041 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     8101 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     8161 aatacgcaaa ccgcctctcc ccgcgcgttg gccgat
//
