LOCUS       sequence-59171-e        6537 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 6537)
  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 6537)
  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 Wnt3-V5
FEATURES             Location/Qualifiers
     source          1..6537
                     /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             2027..2068
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2108..2127)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2152..2200
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2402..2830
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2489..2508)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2699..2720
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2839..2859)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2844..3173
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3024..3159
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3240..4034
                     /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(3294..3313)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3904..3923
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4210..4331
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4247..4266)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4301..4320
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4380..4396)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4380..4396)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4393..4415)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4404..4420
                     /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(4428..4434,4435..4452,4453..4458))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4473..4494
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4611..4628)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4782..5370)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4862..4881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5541..6332,6333..6401))
                     /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     6164..6183
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6402..6506)
                     /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 gccccacctg ctcgggctgc tcctcggcct cctgctcggt ggcaccaggg tcctcgctgg
     1021 ctacccaatt tggtggtccc tggccctggg ccagcagtac acatctctgg gctcacagcc
     1081 cctgctctgc ggctccatcc caggcctggt ccccaagcaa ctgcgcttct gccgcaatta
     1141 catcgagatc atgcccagcg tggccgaggg cgtgaagctg ggcatccagg agtgccagca
     1201 ccagttccgg ggccgccgct ggaactgcac caccatagat gacagcctgg ccatctttgg
     1261 gcccgtcctc gacaaagcca cccgcgagtc ggccttcgtt cacgccatcg cctcggccgg
     1321 cgtggccttc gccgtcaccc gctcctgcgc cgagggcacc tccaccattt gcggctgtga
     1381 ctcgcatcat aaggggccgc ctggcgaagg ctggaagtgg ggcggctgca gcgaggacgc
     1441 tgacttcggc gtgttagtgt ccagggagtt cgcggatgcg cgcgagaaca ggccggacgc
     1501 gcgctcggcc atgaacaagc acaacaacga ggcgggccgc acgactatcc tggaccacat
     1561 gcacctcaaa tgcaagtgcc acgggctgtc gggcagctgt gaggtgaaga cctgctggtg
     1621 ggcgcagcct gacttccgtg ccatcggtga cttcctcaag gacaagtatg acagcgcctc
     1681 ggagatggta gtagagaagc accgtgagtc ccgaggctgg gtggagaccc tccgggccaa
     1741 gtactcgctc ttcaagccac ccacggagag ggacctggtc tactacgaga actcccccaa
     1801 cttttgtgag cccaacccag agacgggttc ctttggcaca agggaccgga cttgcaatgt
     1861 cacctcccac ggcatcgatg gctgcgatct gctctgctgt ggccggggcc acaacacgag
     1921 gacggagaag cggaaggaaa aatgccactg catcttccac tggtgctgct acgtcagctg
     1981 ccaggagtgt attcgcatct acgacgtgca cacctgcaag ggcggaggta agcctatccc
     2041 taaccctctc ctcggtctcg attctacgcg taccggttag taatgagttt aaacggggga
     2101 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     2161 aaagacagaa taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     2221 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     2281 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     2341 gtcggggcgg caggccctgc catagcagat ctgcgcagct ggggctctag ggggtatccc
     2401 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2461 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2521 acgttcgccg gctttccccg tcaagctcta aatcggggca tccctttagg gttccgattt
     2581 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2641 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2701 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2761 taagggattt tggggatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2821 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2881 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2941 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3001 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3061 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3121 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3181 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     3241 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3301 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3361 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3421 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3481 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3541 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3601 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     3661 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     3721 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     3781 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3841 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3901 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3961 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4021 acgagttctt ctgagcggga ctctggggtt cgcgaaatga ccgaccaagc gacgcccaac
     4081 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4141 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4201 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4261 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4321 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4381 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4441 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4501 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4561 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4621 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4681 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4741 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4801 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4861 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4921 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc
     4981 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5041 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5101 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5161 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5221 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5281 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5341 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5401 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5461 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5521 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5581 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5641 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5701 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5761 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5821 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5881 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5941 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6001 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6061 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6121 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6181 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6241 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6301 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6361 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6421 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6481 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
