LOCUS       sequence-193556-        6552 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 6552)
  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 6552)
  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 Wnt2-V5
FEATURES             Location/Qualifiers
     source          1..6552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    677..701
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1808..1849
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1889..1908)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1933..1981
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2183..2611
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2270..2289)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2480..2501
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2620..2640)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2625..2954
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2805..2940
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2867..2886
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3021..3815
                     /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(3075..3094)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3685..3704
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3991..4112
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4028..4047)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4082..4101
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4161..4177)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4161..4177)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4174..4196)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4185..4201
                     /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(4209..4215,4216..4233,4234..4239))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4254..4275
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4392..4409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4563..5151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4643..4662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5322..6113,6114..6182))
                     /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     5945..5964
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6183..6287)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6362..6381)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagttaag ctatcaacaa gtttgtacaa aaaagcaggc tccgcggccg cccccttcac
      721 catgaacgcc cctctcggtg gaatctggct ctggctccct ctgctcttga cctggctcac
      781 ccccgaggtc aactcttcat ggtggtacat gagagctaca ggtggctcct ccagggtgat
      841 gtgcgataat gtgccaggcc tggtgagcag ccagcggcag ctgtgtcacc gacatccaga
      901 tgtgatgcgt gccattagcc agggcgtggc cgagtggaca gcagaatgcc agcaccagtt
      961 ccgccagcac cgctggaatt gcaacaccct ggacagggat cacagccttt ttggcagggt
     1021 cctactccga agtagtcggg aatctgcctt tgtttatgcc atctcctcag ctggagttgt
     1081 atttgccatc accagggcct gtagccaagg agaagtaaaa tcctgttcct gtgatccaaa
     1141 gaagatggga agcgccaagg acagcaaagg catttttgat tggggtggct gcagtgataa
     1201 cattgactat gggatcaaat ttgcccgcgc atttgtggat gcaaaggaaa ggaaaggaaa
     1261 ggatgccaga gccctgatga atcttcacaa caacagagct ggcaggaagg ctgtaaagcg
     1321 gttcttgaaa caagagtgca agtgccacgg ggtgagcggc tcatgtactc tcaggacatg
     1381 ctggctggcc atggccgact tcaggaaaac gggcgattat ctctggagga agtacaatgg
     1441 ggccatccag gtggtcatga accaggatgg cacaggtttc actgtggcta acgagaggtt
     1501 taagaagcca acgaaaaatg acctcgtgta ttttgaaaat tctccagact actgtatcag
     1561 ggaccgagag gcaggctccc tgggtacagc aggccgtgtg tgcaacctga cttcccgggg
     1621 catggacagc tgtgaagtca tgtgctgtgg gagaggctac gacacctccc atgtcacccg
     1681 gatgaccaag tgtgggtgta agttccactg gtgctgcgcc gtgcgctgtc aggactgcct
     1741 ggaagctctg gatgtgcaca catgcaaggc ccccaagaac gctgactgga caaccgctac
     1801 aggcggaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc gtaccggtta
     1861 gtaatgagtt taaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     1921 cccgcgctat gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt
     1981 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     2041 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     2101 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc
     2161 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2221 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2281 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2341 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2401 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2461 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2521 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2581 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     2641 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2701 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2761 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2821 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2881 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2941 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     3001 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3061 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3121 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3181 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3241 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3301 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3361 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3421 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     3481 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     3541 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     3601 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     3661 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     3721 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     3781 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgcgaaatg
     3841 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     3901 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3961 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4021 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4081 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4141 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4201 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4261 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4321 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4381 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4441 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4501 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4561 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4621 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4681 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4741 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4801 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4861 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4921 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4981 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5041 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5101 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5161 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5221 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5281 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5341 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5401 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5461 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5521 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5581 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5641 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5701 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5761 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5821 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5881 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5941 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6001 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6061 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6121 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6181 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6241 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6301 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     6361 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     6421 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     6481 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     6541 gatatacgcg tt
//
