LOCUS       sequence-59172-e        6528 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 6528)
  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 6528)
  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 Wnt3A-V5
FEATURES             Location/Qualifiers
     source          1..6528
                     /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             2018..2059
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2099..2118)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2143..2191
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2393..2821
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2480..2499)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2690..2711
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2830..2850)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2835..3164
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3015..3150
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3077..3096
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3231..4025
                     /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(3285..3304)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3895..3914
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4201..4322
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4238..4257)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4292..4311
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4371..4387)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4371..4387)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4384..4406)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4395..4411
                     /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(4419..4425,4426..4443,4444..4449))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4464..4485
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4602..4619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4773..5361)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4853..4872)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5532..6323,6324..6392))
                     /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     6155..6174
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6393..6497)
                     /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 tcaccatggc
      961 cccactcgga tacttcttac tcctctgcag cctgaagcag gctctgggca gctacccgat
     1021 ctggtggtcg ctggctgttg ggccacagta ttcctccctg ggctcgcagc ccatcctgtg
     1081 tgccagcatc ccgggcctgg tccccaagca gctccgcttc tgcaggaact acgtggagat
     1141 catgcccagc gtggccgagg gcatcaagat tggcatccag gagtgccagc accagttccg
     1201 cggccgccgg tggaactgca ccaccgtcca cgacagcctg gccatcttcg ggcccgtgct
     1261 ggacaaagct accagggagt cggcctttgt ccacgccatt gcctcagccg gtgtggcctt
     1321 tgcagtgaca cgctcatgtg cagaaggcac ggccgccatc tgtggctgca gcagccgcca
     1381 ccagggctca ccaggcaagg gctggaagtg gggtggctgt agcgaggaca tcgagtttgg
     1441 tgggatggtg tctcgggagt tcgccgacgc ccgggagaac cggccagatg cccgctcagc
     1501 catgaaccgc cacaacaacg aggctgggcg ccaggccatc gccagccaca tgcacctcaa
     1561 gtgcaagtgc cacgggctgt cgggcagctg cgaggtgaag acatgctggt ggtcgcaacc
     1621 cgacttccgc gccatcggtg acttcctcaa ggacaagtac gacagcgcct cggagatggt
     1681 ggtggagaag caccgggagt cccgcggctg ggtggagacc ctgcggccgc gctacaccta
     1741 cttcaaggtg cccacggagc gcgacctggt ctactacgag gcctcgccca acttctgcga
     1801 gcccaaccct gagacgggct ccttcggcac gcgcgaccgc acctgcaacg tcagctcgca
     1861 cggcatcgac ggctgcgacc tgctgtgctg cggccgcggc cacaacgcgc gagcggagcg
     1921 gcgccgggag aagtgccgct gcgtgttcca ctggtgctgc tacgtcagct gccaggagtg
     1981 cacgcgcgtc tacgacgtgc acacctgcaa gggcggaggt aagcctatcc ctaaccctct
     2041 cctcggtctc gattctacgc gtaccggtta gtaatgagtt taaacggggg aggctaactg
     2101 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     2161 ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     2221 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     2281 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     2341 gcaggccctg ccatagcaga tctgcgcagc tggggctcta gggggtatcc ccacgcgccc
     2401 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2461 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2521 ggctttcccc gtcaagctct aaatcggggc atccctttag ggttccgatt tagtgcttta
     2581 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2641 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2701 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2761 ttggggattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2821 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2881 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     2941 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3001 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3061 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3121 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     3181 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     3241 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3301 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3361 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     3421 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3481 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3541 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     3601 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     3661 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     3721 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     3781 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     3841 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     3901 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     3961 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4021 tctgagcggg actctggggt tcgcgaaatg accgaccaag cgacgcccaa cctgccatca
     4081 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4141 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     4201 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4261 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4321 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4381 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4441 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4501 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4561 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4621 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4681 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4741 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4801 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4861 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4921 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt
     4981 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5041 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5101 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5161 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     5221 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5281 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5341 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5401 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5461 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5521 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5581 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5641 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5701 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5761 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5821 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5881 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5941 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6001 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6061 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6121 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6181 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6241 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6301 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6361 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6421 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6481 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
