LOCUS       sequence-59156-e        6567 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 6567)
  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 6567)
  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 Wnt16-V5
FEATURES             Location/Qualifiers
     source          1..6567
                     /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             2057..2098
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2138..2157)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2182..2230
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2432..2860
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2519..2538)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2729..2750
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2869..2889)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2874..3203
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3054..3189
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3116..3135
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3270..4064
                     /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(3324..3343)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3934..3953
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4240..4361
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4277..4296)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4331..4350
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4410..4426)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4410..4426)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4423..4445)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4434..4450
                     /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(4458..4464,4465..4482,4483..4488))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4503..4524
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4641..4658)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4812..5400)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4892..4911)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5571..6362,6363..6431))
                     /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     6194..6213
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6432..6536)
                     /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 tcaccatgga
      961 cagggcggcg ctcctgggac tggcccgctt gtgcgcgctg tgggcagccc tgctcgtgct
     1021 gttcccctac ggagcccaag gaaactggat gtggttgggc attgcctcct tcggggttcc
     1081 agagaagctg ggctgcgcca atttgccgct gaacagccgc cagaaggagc tgtgcaagag
     1141 gaaaccgtac ctgctgccga gcatccgaga gggcgcccgg ctgggcattc aggagtgcgg
     1201 gagccagttc agacacgaga gatggaactg catgatcacc gccgccgcca ctaccgcccc
     1261 gatgggcgcc agccccctct ttggctacga gctgagcagc ggcaccaaag agacagcatt
     1321 tatttatgct gtgatggctg caggcctggt gcattctgtg accaggtcat gcagtgcagg
     1381 caacatgaca gagtgttcct gtgacaccac cttgcagaac ggcggctcag caagtgaagg
     1441 ctggcactgg gggggctgct ccgatgatgt ccagtatggc atgtggttca gcagaaagtt
     1501 cctagatttc cccatcggaa acaccacggg caaagaaaac aaagtactat tagcaatgaa
     1561 cctacataac aatgaagctg gaaggcaggc tgtcgccaag ttgatgtcag tagactgccg
     1621 ctgccacgga gtttccggct cctgtgctgt gaaaacatgc tggaaaacca tgtcttcttt
     1681 tgaaaagatt ggccatttgt tgaaggataa atatgaaaac agtatccaga tatcagacaa
     1741 aacaaagagg aaaatgcgca ggagagaaaa agatcagagg aaaataccaa tccataagga
     1801 tgatctgctc tatgttaata agtctcccaa ctactgtgta gaagataaga aactgggaat
     1861 cccagggaca caaggcagag aatgcaaccg tacatcagag ggtgcagatg gctgcaacct
     1921 cctctgctgt ggccgaggtt acaacaccca tgtggtcagg cacgtggaga ggtgtgagtg
     1981 taagttcatc tggtgctgct atgtccgttg caggaggtgt gaaagcatga ctgatgtcca
     2041 cacttgcaag ggcggaggta agcctatccc taaccctctc ctcggtctcg attctacgcg
     2101 taccggttag taatgagttt aaacggggga ggctaactga aacacggaag gagacaatac
     2161 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac gggtgttggg
     2221 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     2281 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     2341 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcagat
     2401 ctgcgcagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2461 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2521 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2581 aatcggggca tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2641 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2701 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2761 aaccctatct cggtctattc ttttgattta taagggattt tggggatttc ggcctattgg
     2821 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     2881 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2941 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3001 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3061 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3121 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     3181 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     3241 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3301 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3361 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3421 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3481 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3541 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3601 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3661 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3721 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3781 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3841 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3901 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3961 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4021 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4081 cgcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4141 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4201 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4261 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4321 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4381 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4441 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4501 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4561 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4621 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4681 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4741 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4801 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4861 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4921 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4981 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     5041 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5101 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5161 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5221 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5281 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5341 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5401 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5461 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5521 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5581 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5641 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5701 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5761 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5821 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5881 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5941 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6001 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6061 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6121 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6181 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6241 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6301 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6361 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6421 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6481 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6541 tttccccgaa aagtgccacc tgacgtc
//
