LOCUS       sequence-59161-e        6543 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 6543)
  AUTHORS   MacDonald BT, Hien A, Zhang X, Iranloye O, Virshup DM, Waterman ML, 
            He X
  TITLE     Disulfide Bond Requirements for Active Wnt Ligands.
  JOURNAL   J Biol Chem. 2014 May 19. pii: jbc.M114.575027.
  PUBMED    24841207
REFERENCE   2  (bases 1 to 6543)
  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 
            Chem. 2014 May 19. pii: jbc.M114.575027."
COMMENT     Sequence Label: Active Wnt9B-V5
FEATURES             Location/Qualifiers
     source          1..6543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    911..935
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2033..2074
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2114..2133)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2158..2206
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2408..2836
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2495..2514)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2705..2726
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2845..2865)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2850..3179
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3030..3165
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3092..3111
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3246..4040
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3300..3319)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3910..3929
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4216..4337
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4253..4272)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4307..4326
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4386..4402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4386..4402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4399..4421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4410..4426
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4434..4440,4441..4458,4459..4464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4479..4500
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4617..4634)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4788..5376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4868..4887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5547..6338,6339..6407))
                     /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     6170..6189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6408..6512)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagctatca acaagtttgt acaaaaaagc aggctccgcg gccgccccct tcaccatgcg
      961 ccccccgccc gcgctggccc tggccgggct ctgcctgctg gcgctgcccg ccgccgccgc
     1021 ctcctacttc ggcctgaccg ggcgggaagt cctgacgccc ttcccaggat tgggcactgc
     1081 ggcagccccg gcacagggcg gggcccacct gaagcagtgt gacctgctga agctgtcccg
     1141 gcggcagaag cagctctgcc ggagggagcc cggcctggct gagaccctga gggatgctgc
     1201 gcacctcggc ctgcttgagt gccagtttca gttccggcat gagcgctgga actgtagcct
     1261 ggagggcagg acgggcctgc tcaagagagg cttcaaagag acagctttcc tgtacgcggt
     1321 gtcctctgcc gccctcaccc acaccctggc ccgggcctgc agcgctgggc gcatggagcg
     1381 ctgcacctgt gatgactctc cggggctgga gagccggcag gcctggcagt ggggcgtgtg
     1441 cggtgacaac ctcaagtaca gcaccaagtt tctgagcaac ttcctggggt ccaagagagg
     1501 aaacaaggac ctgcgggcac gggcagacgc ccacaatacc cacgtgggca tcaaggctgt
     1561 gaagagtggc ctcaggacca cgtgtaagtg ccatggcgta tcaggctcct gtgccgtgcg
     1621 cacctgctgg aagcagctct ccccgttccg tgagacgggc caggtgctga aactgcgcta
     1681 tgactcggct gtcaaggtgt ccagtgccac caatgaggcc ttgggccgcc tagagctgtg
     1741 ggcccctgcc aggcagggca gcctcaccaa aggcctggcc ccaaggtctg gggacctggt
     1801 gtacatggag gactcaccca gcttctgccg gcccagcaag tactcacctg gcacagcagg
     1861 tagggtgtgc tcccgggagg ccagctgcag cagcctgtgc tgcgggcggg gctatgacac
     1921 ccagagccgc ctggtggcct tctcctgcca ctgccaggtg cagtggtgct gctacgtgga
     1981 gtgccagcaa tgtgtgcagg aggagcttgt gtacacctgc aagcacggcg gaggtaagcc
     2041 tatccctaac cctctcctcg gtctcgattc tacgcgtacc ggttagtaat gagtttaaac
     2101 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     2161 caataaaaag acagaataaa acgcacgggt gttgggtcgt ttgttcataa acgcggggtt
     2221 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
     2281 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
     2341 gccaacgtcg gggcggcagg ccctgccata gcagatctgc gcagctgggg ctctaggggg
     2401 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2461 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2521 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc ggggcatccc tttagggttc
     2581 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2641 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2701 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2761 gatttataag ggattttggg gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2821 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     2881 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     2941 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3001 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3061 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     3121 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3181 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     3241 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3301 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3361 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3421 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3481 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3541 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3601 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     3661 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     3721 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     3781 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     3841 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     3901 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     3961 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4021 ttcttgacga gttcttctga gcgggactct ggggttcgcg aaatgaccga ccaagcgacg
     4081 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     4141 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     4201 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4261 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4321 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     4381 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4441 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4501 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4561 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4621 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4681 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4741 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4801 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4861 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4921 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4981 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5041 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5101 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5161 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5221 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5281 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5341 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5401 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5461 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5521 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5581 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5641 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5701 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5761 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5821 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5881 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5941 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6001 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6061 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6121 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6181 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6241 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6301 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6361 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6421 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6481 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6541 gtc
//
