LOCUS       sequence-59157-e        6534 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 6534)
  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 6534)
  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 Wnt11-V5
FEATURES             Location/Qualifiers
     source          1..6534
                     /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             2024..2065
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2105..2124)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2149..2197
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2399..2827
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2486..2505)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2696..2717
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2836..2856)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2841..3170
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3021..3156
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3083..3102
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3237..4031
                     /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(3291..3310)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3901..3920
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4207..4328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4244..4263)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4298..4317
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4377..4393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4377..4393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4390..4412)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4401..4417
                     /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(4425..4431,4432..4449,4450..4455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4470..4491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4608..4625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4779..5367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4859..4878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5538..6329,6330..6398))
                     /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     6161..6180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6399..6503)
                     /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 tcaccatgag
      961 ggcgcggccg caggtctgcg aggcgctgct cttcgccctg gcgctccaga ccggcgtgtg
     1021 ctatggcatc aagtggctgg cgctgtccaa gacaccatcg gccctggcac tgaaccagac
     1081 gcaacactgc aagcagctgg agggtctggt gtctgcacag gtgcagctgt gccgcagcaa
     1141 cctggagctc atgcacacgg tggtgcacgc cgcccgcgag gtcatgaagg cctgtcgccg
     1201 ggcctttgcc gacatgcgct ggaactgctc ctccattgag ctcgccccca actatttgct
     1261 tgacctggag agagggaccc gggagtcggc cttcgtgtat gcgctgtcgg ccgccgccat
     1321 cagccacgcc atcgcccggg cctgcacctc cggcgacctg cccggctgct cctgcggccc
     1381 cgtcccaggt gagccacccg ggcccgggaa ccgctgggga ggatgtgcgg acaacctcag
     1441 ctacgggctc ctcatggggg ccaagttttc cgatgctcct atgaaggtga aaaaaacagg
     1501 atcccaagcc aataaactga tgcgtctaca caacagtgaa gtggggagac aggctctgcg
     1561 cgcctctctg gaaatgaagt gtaagtgcca tggggtgtct ggctcctgct ccatccgcac
     1621 ctgctggaag gggctgcagg agctgcagga tgtggctgct gacctcaaga cccgatacct
     1681 gtcggccacc aaggtagtgc accgacccat gggcacccgc aagcacctgg tgcccaagga
     1741 cctggatatc cggcctgtga aggactcgga actcgtctat ctgcagagct cacctgactt
     1801 ctgcatgaag aatgagaagg tgggctccca cgggacacaa gacaggcagt gcaacaagac
     1861 atccaacgga agcgacagct gcgaccttat gtgctgcggg cgtggctaca acccctacac
     1921 agaccgcgtg gtcgagcggt gccactgtaa gtaccactgg tgctgctacg tcacctgccg
     1981 caggtgtgag cgtaccgtgg agcgctatgt ctgcaagggc ggaggtaagc ctatccctaa
     2041 ccctctcctc ggtctcgatt ctacgcgtac cggttagtaa tgagtttaaa cgggggaggc
     2101 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     2161 gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     2221 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     2281 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     2341 ggggcggcag gccctgccat agcagatctg cgcagctggg gctctagggg gtatccccac
     2401 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2461 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2521 ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt ccgatttagt
     2581 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2641 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2701 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2761 gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2821 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2881 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2941 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3001 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3061 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     3121 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     3181 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     3241 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3301 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3361 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     3421 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3481 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3541 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3601 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3661 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3721 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     3781 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3841 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3901 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3961 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4021 agttcttctg agcgggactc tggggttcgc gaaatgaccg accaagcgac gcccaacctg
     4081 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4141 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4201 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4261 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4321 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4381 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4441 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4621 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4681 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4741 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4801 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4861 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4921 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     4981 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5041 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5101 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5161 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5221 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5281 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5341 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5401 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5461 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5521 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5581 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5641 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5701 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5761 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5821 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5881 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5941 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6001 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6061 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6121 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6181 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6241 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6301 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6361 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6421 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6481 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
