LOCUS       sequence-59158-e        6639 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 6639)
  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 6639)
  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 Wnt10B-V5
FEATURES             Location/Qualifiers
     source          1..6639
                     /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             2129..2170
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2210..2229)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2254..2302
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2504..2932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2591..2610)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2801..2822
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2941..2961)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2946..3275
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3126..3261
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3188..3207
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3342..4136
                     /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(3396..3415)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4006..4025
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4312..4433
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4349..4368)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4403..4422
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4482..4498)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4482..4498)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4495..4517)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4506..4522
                     /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(4530..4536,4537..4554,4555..4560))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4575..4596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4713..4730)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4884..5472)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4964..4983)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5643..6434,6435..6503))
                     /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     6266..6285
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6504..6608)
                     /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 tcaccatgct
      961 ggaggagccc cggccgcggc ctccgccctc gggcctcgcg ggtctcctgt tcctggcgtt
     1021 gtgcagtcgg gctctaagca atgagattct gggcctgaag ttgcctggcg agccgccgct
     1081 gacggccaac accgtgtgct tgacgctgtc cggcctgagc aagcggcagc taggcctgtg
     1141 cctgcgcaac cccgacgtga cggcgtccgc gcttcagggt ctgcacatcg cggtccacga
     1201 gtgtcagcac cagctgcgcg accagcgctg gaactgctcc gcgcttgagg gcggcggccg
     1261 cctgccgcac cacagcgcca tcctcaagcg cggtttccga gaaagtgctt tttccttctc
     1321 catgctggct gctggggtca tgcacgcagt agccacggcc tgcagcctgg gcaagctggt
     1381 gagctgtggc tgtggctgga agggcagtgg tgagcaggat cggctgaggg ccaaactgct
     1441 gcagctgcag gcactgtccc gaggcaagag tttcccccac tctctgccca gccctggccc
     1501 tggctcaagc cccagccctg gcccccagga cacatgggaa tggggtggct gtaaccatga
     1561 catggacttt ggagagaagt tctctcggga tttcttggat tccagggaag ctccccggga
     1621 catccaggca cgaatgcgaa tccacaacaa cagggtgggg cgccaggtgg taactgaaaa
     1681 cctgaagcgg aaatgcaagt gtcatggcac atcaggcagc tgccagttca agacatgctg
     1741 gagggcggcc ccagagttcc gggcagtggg ggcggcgttg agggagcggc tgggccgggc
     1801 catcttcatt gatacccaca accgcaattc tggagccttc cagccccgtc tgcgtccccg
     1861 tcgcctctca ggagagctgg tctactttga gaagtctcct gacttctgtg agcgagaccc
     1921 cactatgggc tccccaggga caaggggccg ggcctgcaac aagaccagcc gcctgttgga
     1981 tggctgtggc agcctgtgct gtggccgtgg gcacaacgtg ctccggcaga cacgagttga
     2041 gcgctgccat tgccgcttcc actggtgctg ctatgtgctg tgtgatgagt gcaaggttac
     2101 agagtgggtg aatgtgtgta agggcggagg taagcctatc cctaaccctc tcctcggtct
     2161 cgattctacg cgtaccggtt agtaatgagt ttaaacgggg gaggctaact gaaacacgga
     2221 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     2281 acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     2341 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     2401 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     2461 gccatagcag atctgcgcag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2521 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2581 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2641 cgtcaagctc taaatcgggg catcccttta gggttccgat ttagtgcttt acggcacctc
     2701 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2761 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2821 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttggggatt
     2881 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     2941 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3001 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3061 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3121 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3181 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3241 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3301 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3361 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3421 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3481 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3541 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3601 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3661 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3721 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     3781 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     3841 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     3901 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     3961 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4021 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4081 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4141 gactctgggg ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4201 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4261 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4321 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4381 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4441 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4501 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4561 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4621 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4681 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4741 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4801 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4861 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4921 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4981 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5041 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
     5101 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5161 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5221 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5281 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5341 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5401 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5461 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5521 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5581 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5641 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5701 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5761 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5821 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5881 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5941 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6001 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6061 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6121 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6181 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6241 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6301 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6361 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6421 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6481 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6541 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6601 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
