LOCUS       sequence-59164-e        6525 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 6525)
  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 6525)
  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 Wnt8A-V5
FEATURES             Location/Qualifiers
     source          1..6525
                     /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             1513..1524
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             2015..2056
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2096..2115)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2140..2188
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2390..2818
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2477..2496)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2687..2708
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2827..2847)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2832..3161
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3012..3147
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3074..3093
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3228..4022
                     /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(3282..3301)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3892..3911
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4198..4319
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4235..4254)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4289..4308
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4368..4384)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4368..4384)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4381..4403)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4392..4408
                     /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(4416..4422,4423..4440,4441..4446))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4461..4482
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4599..4616)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4770..5358)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4850..4869)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5529..6320,6321..6389))
                     /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     6152..6171
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6390..6494)
                     /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 tcaccatggg
      961 gaacctgttt atgctctggg cagctctggg catatgctgt gctgcattca gtgcctctgc
     1021 ctggtcagtg aacaatttcc tgataacagg tcccaaggcc tatctgacct acacgactag
     1081 tgtggccttg ggtgcccaga gtggcatcga ggagtgcaag ttccagtttg cttgggaacg
     1141 ctggaactgc cctgaaaatg ctcttcagct ctccacccac aacaggctga gaagtgctac
     1201 cagagagact tccttcatac atgctatcag ctctgctgga gtcatgtaca tcatcaccaa
     1261 gaactgtagc atgggtgact tcgaaaactg tggctgtgat gggtcaaaca atggaaaaac
     1321 aggaggccat ggctggatct ggggaggctg cagcgacaat gtggaatttg gggaaaggat
     1381 ctccaaactc tttgtggaca gtttggagaa ggggaaggat gccagagccc tgatgaatct
     1441 tcacaacaac agggccggca gactggcagt gagagccacc atgaaaagga catgcaaatg
     1501 tcatggcatc tctgggagct gcagcataca gacatgctgg ctgcagctgg ctgagttccg
     1561 ggagatggga gactacctaa aggccaagta tgaccaggcg ctgaaaattg aaatggataa
     1621 gcggcagctg agagctggga acagcgccga gggccactgg gtgcccgctg aggccttcct
     1681 tcctagcgca gaggcggaac tgatcttttt agaggaatca ccagattact gtacctgcaa
     1741 ttccagcctg ggcatctatg gcacagaggg tcgtgagtgc ctacagaaca gccacaacac
     1801 atccaggtgg gagcgacgta gctgtgggcg cctgtgcact gagtgtgggc tgcaggtgga
     1861 agagaggaaa actgaggtca taagcagctg taactgcaaa ttccagtggt gctgtacggt
     1921 caagtgtgac cagtgtaggc atgtggtgag caagtattac tgcgcacgct ccccaggcag
     1981 tgcccagtcc ctgggtaagg gcagtgccgg cggaggtaag cctatcccta accctctcct
     2041 cggtctcgat tctacgcgta ccggttagta atgagtttaa acgggggagg ctaactgaaa
     2101 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     2161 aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     2221 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     2281 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     2341 ggccctgcca tagcagatct gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt
     2401 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2461 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2521 tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag tgctttacgg
     2581 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2641 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2701 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2761 gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     2821 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2881 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2941 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3001 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3061 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     3121 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     3181 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3241 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3301 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3361 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     3421 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3481 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3541 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3601 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3661 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3721 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     3781 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     3841 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     3901 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     3961 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4021 gagcgggact ctggggttcg cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4081 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4141 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     4201 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4261 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4321 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4381 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4441 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4501 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4561 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4621 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4681 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4741 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4801 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4861 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4921 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     4981 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5041 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5101 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5161 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     5221 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5281 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5341 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5401 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5461 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5521 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5581 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5641 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5701 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5761 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5821 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5881 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5941 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6001 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6061 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6121 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6181 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6241 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6301 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6361 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6421 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6481 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
