LOCUS       sequence-59169-e        6612 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 6612)
  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 6612)
  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 Wnt5A-V5
FEATURES             Location/Qualifiers
     source          1..6612
                     /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             2102..2143
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2183..2202)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2227..2275
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2477..2905
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2564..2583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2774..2795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2914..2934)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2919..3248
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3099..3234
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3161..3180
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3315..4109
                     /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(3369..3388)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3979..3998
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4285..4406
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4322..4341)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4376..4395
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4455..4471)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4455..4471)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4468..4490)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4479..4495
                     /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(4503..4509,4510..4527,4528..4533))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4548..4569
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4686..4703)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4857..5445)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4937..4956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5616..6407,6408..6476))
                     /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     6239..6258
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6477..6581)
                     /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 tcaccatgaa
      961 gaagtccatt ggaatattaa gcccaggagt tgctttgggg atggctggaa gtgcaatgtc
     1021 ttccaagttc ttcctagtgg ctttggccat atttttctcc ttcgcccagg ttgtaattga
     1081 agccaattct tggtggtcgc taggtatgaa taaccctgtt cagatgtcag aagtatatat
     1141 tataggagca cagcctctct gcagccaact ggcaggactt tctcaaggac agaagaaact
     1201 gtgccacttg tatcaggacc acatgcagta catcggagaa ggcgcgaaga caggcatcaa
     1261 agaatgccag tatcaattcc gacatcgaag gtggaactgc agcactgtgg ataacacctc
     1321 tgtttttggc agggtgatgc agataggcag ccgcgagacg gccttcacat acgcggtgag
     1381 cgcagcaggg gtggtgaacg ccatgagccg ggcgtgccgc gagggcgagc tgtccacctg
     1441 cggctgcagc cgcgccgcgc gccccaagga cctgccgcgg gactggctct ggggcggctg
     1501 cggcgacaac atcgactatg gctaccgctt tgccaaggag ttcgtggacg cccgcgagcg
     1561 ggagcgcatc cacgccaagg gctcctacga gagtgctcgc atcctcatga acctgcacaa
     1621 caacgaggcc ggccgcagga cggtgtacaa cctggctgat gtggcctgca agtgccatgg
     1681 ggtgtccggc tcatgtagcc tgaagacatg ctggctgcag ctggcagact tccgcaaggt
     1741 gggtgatgcc ctgaaggaga agtacgacag cgcggcggcc atgcggctca acagccgggg
     1801 caagttggta caggtcaaca gccgcttcaa ctcgcccacc acacaagacc tggtctacat
     1861 cgaccccagc cctgactact gcgtgcgcaa tgagagcacc ggctcgctgg gcacgcaggg
     1921 ccgcctgtgc aacaagacgt cggagggcat ggatggctgc gagctcatgt gctgcggccg
     1981 tggctacgac cagttcaaga ccgtgcagac ggagcgctgc cactgcaagt tccactggtg
     2041 ctgctacgtc aagtgcaaga agtgcacgga gatcgtggac cagtttgtgt gcaagggcgg
     2101 aggtaagcct atccctaacc ctctcctcgg tctcgattct acgcgtaccg gttagtaatg
     2161 agtttaaacg ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     2221 ctatgacggc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa
     2281 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     2341 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     2401 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg cagctggggc
     2461 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2521 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2581 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct
     2641 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2701 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2761 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2821 tattcttttg atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg
     2881 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     2941 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3001 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3061 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3121 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3181 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3241 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     3301 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3361 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3421 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3481 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3541 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3601 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3661 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3721 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3781 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3841 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3901 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3961 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4021 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4081 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgcga aatgaccgac
     4141 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4201 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4261 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4321 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4381 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4441 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4501 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4561 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4621 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4681 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4741 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4801 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4861 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4921 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4981 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5041 gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5101 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5161 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5221 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5281 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5341 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5401 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5461 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5521 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5581 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5641 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5701 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5761 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5821 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5881 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5941 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6001 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6061 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6121 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6181 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6241 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6301 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6361 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6421 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6481 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6541 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6601 ccacctgacg tc
//
