LOCUS       sequence-59165-e        6519 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6519)
  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 6519)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 Wnt7B-V5
FEATURES             Location/Qualifiers
     source          1..6519
                     /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             2009..2050
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2090..2109)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2134..2182
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2384..2812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2471..2490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2681..2702
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2821..2841)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2826..3155
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3006..3141
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3068..3087
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3222..4016
                     /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(3276..3295)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3886..3905
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4192..4313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4229..4248)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4283..4302
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4362..4378)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4362..4378)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4375..4397)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4386..4402
                     /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(4410..4416,4417..4434,4435..4440))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4455..4476
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4593..4610)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4764..5352)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4844..4863)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5523..6314,6315..6383))
                     /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     6146..6165
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6384..6488)
                     /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 tcaccatgca
      961 cagaaacttt cgcaagtgga ttttctacgt gtttctctgc tttggcgtcc tgtacgtgaa
     1021 gctcggagca ctgtcatccg tggtggccct gggagccaac atcatctgca acaagattcc
     1081 tggcctagcc ccgcggcagc gtgccatctg ccagagtcgg cccgatgcca tcattgtgat
     1141 tggggagggg gcgcagatgg gcatcaacga gtgccagtac cagttccgct tcggacgctg
     1201 gaactgctct gccctcggcg agaagaccgt cttcgggcaa gagctccgag tagggagccg
     1261 tgaggctgcc ttcacgtacg ccatcaccgc ggctggcgtg gcgcacgccg tcaccgctgc
     1321 ctgcagccaa gggaacctga gcaactgcgg ctgcgaccgc gagaagcagg gctactacaa
     1381 ccaagccgag ggctggaagt ggggcggctg ctcggccgac gtgcgttacg gcatcgactt
     1441 ctcccggcgc ttcgtggacg ctcgggagat caagaagaac gcgcggcgcc tcatgaacct
     1501 gcataacaat gaggccggca ggaaggttct agaggaccgg atgcagctgg agtgcaagtg
     1561 ccacggcgtg tctggctcct gcaccaccaa aacctgctgg accacgctgc ccaagttccg
     1621 agaggtgggc cacctgctga aggagaagta caacgcggcc gtgcaggtgg aggtggtgcg
     1681 ggccagccgt ctgcggcagc ccaccttcct gcgcatcaaa cagctgcgca gctatcagaa
     1741 gcccatggag acagacctgg tgtacattga gaagtcgccc aactactgcg aggaggacgc
     1801 ggccacgggc agcgtgggca cgcagggccg tctctgcaac cgcacgtcgc ccggcgcgga
     1861 cggctgtgac accatgtgct gcggccgagg ctacaacacc caccagtaca ccaaggtgtg
     1921 gcagtgcaac tgcaaattcc actggtgctg cttcgtcaag tgcaacacct gcagcgagcg
     1981 caccgaggtc ttcacctgca agggcggagg taagcctatc cctaaccctc tcctcggtct
     2041 cgattctacg cgtaccggtt agtaatgagt ttaaacgggg gaggctaact gaaacacgga
     2101 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     2161 acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     2221 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     2281 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     2341 gccatagcag atctgcgcag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2401 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2461 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2521 cgtcaagctc taaatcgggg catcccttta gggttccgat ttagtgcttt acggcacctc
     2581 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2641 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2701 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttggggatt
     2761 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     2821 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2881 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     2941 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3001 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3061 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3121 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3181 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3241 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3301 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3361 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3421 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3481 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3541 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3601 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     3661 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     3721 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     3781 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     3841 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     3901 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     3961 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4021 gactctgggg ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4081 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4141 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4201 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4261 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4321 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4381 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4441 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4501 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4561 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4621 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4681 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4741 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4801 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4861 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4921 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
     4981 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5041 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5101 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5161 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5221 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5281 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5341 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5401 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5461 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5521 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5581 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5641 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5701 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5761 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5821 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5881 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5941 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6001 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6061 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6121 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6181 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6241 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6301 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6361 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6421 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6481 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
