LOCUS       sequence-59168-e        6549 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 6549)
  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 6549)
  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 Wnt5B-V5
FEATURES             Location/Qualifiers
     source          1..6549
                     /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             2039..2080
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2120..2139)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2164..2212
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2414..2842
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2501..2520)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2711..2732
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2851..2871)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2856..3185
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3036..3171
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3098..3117
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3252..4046
                     /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(3306..3325)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3916..3935
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4222..4343
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4259..4278)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4313..4332
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4392..4408)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4392..4408)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4405..4427)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4416..4432
                     /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(4440..4446,4447..4464,4465..4470))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4485..4506
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4623..4640)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4794..5382)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4874..4893)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5553..6344,6345..6413))
                     /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     6176..6195
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6414..6518)
                     /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 tcaccatgcc
      961 cagcctgctg ctgctgttca cggctgctct gctgtccagc tgggctcagc ttctgacaga
     1021 cgccaactcc tggtggtcat tagctttgaa cccggtgcag agacccgaga tgtttatcat
     1081 cggtgcccag cccgtgtgca gtcagcttcc cgggctctcc cctggccaga ggaagctgtg
     1141 ccaattgtac caggagcaca tggcctacat aggggaggga gccaagactg gcatcaagga
     1201 atgccagcac cagttccggc agcggcggtg gaattgcagc acagcggaca acgcatctgt
     1261 ctttgggaga gtcatgcaga taggcagccg agagaccgcc ttcacccacg cggtgagcgc
     1321 cgcgggcgtg gtcaacgcca tcagccgggc ctgccgcgag ggcgagctct ccacctgcgg
     1381 ctgcagccgg acggcgcggc ccaaggacct gccccgggac tggctgtggg gcggctgtgg
     1441 ggacaacgtg gagtacggct accgcttcgc caaggagttt gtggatgccc gggagcgaga
     1501 gaagaacttt gccaaaggat cagaggagca gggccgggtg ctcatgaacc tgcaaaacaa
     1561 cgaggccggt cgcagggctg tgtataagat ggcagacgta gcctgcaaat gccacggcgt
     1621 ctcggggtcc tgcagcctca agacctgctg gctgcagctg gccgagttcc gcaaggtcgg
     1681 ggaccggctg aaggagaagt acgacagcgc ggccgccatg cgcgtcaccc gcaagggccg
     1741 gctggagctg gtcaacagcc gcttcaccca gcccaccccg gaggacctgg tctatgtgga
     1801 ccccagcccc gactactgcc tgcgcaacga gagcacgggc tccctgggca cgcagggccg
     1861 cctctgcaac aagacctcgg agggcatgga tggctgtgag ctcatgtgct gcgggcgtgg
     1921 ctacaaccag ttcaagagcg tgcaggtgga gcgctgccac tgcaagttcc actggtgctg
     1981 cttcgtcagg tgtaagaagt gcacggagat cgtggaccag tacatctgta aaggcggagg
     2041 taagcctatc cctaaccctc tcctcggtct cgattctacg cgtaccggtt agtaatgagt
     2101 ttaaacgggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     2161 tgacggcaat aaaaagacag aataaaacgc acgggtgttg ggtcgtttgt tcataaacgc
     2221 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa
     2281 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg
     2341 ctcgcagcca acgtcggggc ggcaggccct gccatagcag atctgcgcag ctggggctct
     2401 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2461 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2521 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg catcccttta
     2581 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2641 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2701 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2761 tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa tgagctgatt
     2821 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     2881 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2941 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3001 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3061 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3121 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3181 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     3241 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3301 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3361 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3421 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3481 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3541 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3601 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3661 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3721 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     3781 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     3841 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     3901 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     3961 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4021 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgcgaaat gaccgaccaa
     4081 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4141 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4201 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4261 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4321 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4381 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4441 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4501 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4561 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4621 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4681 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4741 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4801 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4861 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4921 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4981 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5041 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5101 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5161 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5221 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5281 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5341 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5401 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5461 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5521 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5581 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5641 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5701 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5761 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5821 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5881 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5941 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6001 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6061 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6121 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6181 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6241 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6301 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6361 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6421 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6481 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6541 cctgacgtc
//
