LOCUS       sequence-329626-        6932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eCFP-VAMP8(G7-R67)-mVenus in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6932)
  AUTHORS   Blum TR, Liu H, Packer MS, Xiong X, Lee PG, Zhang S, Richter M, 
            Minasov G, Satchell KJF, Dong M, Liu DR
  TITLE     Phage-assisted evolution of botulinum neurotoxin proteases with 
            reprogrammed specificity.
  JOURNAL   Science. 2021 Feb 19;371(6531):803-810. doi: 
            10.1126/science.abf5972.
  PUBMED    33602850
REFERENCE   2  (bases 1 to 6932)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2021-02-19- 19"; volume: "371"; 
            issue: "6531"; pages: "803-810"
COMMENT     Sequence Label: pET-CVm-VAMP8
FEATURES             Location/Qualifiers
     source          1..6932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(158..868,869..871,872..874))
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(200..221)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     549..568
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     808..829
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(join(881..895,896..904))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             complement(1835..1864)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     RBS             1873..1895
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1910..1934
                     /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)."
     primer_bind     complement(1934..1953)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1935..1953)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2037..2056)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2262..2339
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2340..3422
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2359..2378)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4231..4422
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4416..4438)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4524..4663
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4579..4598
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4678..4695)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4849..5437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4929..4948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5548..5567)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5559..6374
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5559..6374
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5632..5651)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6466..6921)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6603..6624)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6815..6834
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctt gtacagctcg tccatgccga
      181 gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg
      241 ggtctttgct cagggcggac tggtagctca ggtagtggtt gtcgggcagc agcacggggc
      301 cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgccg ccgtcctcga
      361 tgttgtggcg gatcttgaag ttggccttga tgccgttctt ctgcttgtcg gcggtgatat
      421 agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct
      481 tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct
      541 cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc
      601 cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcgggcga
      661 agcactgcag gccgtagccc agggtggtca cgagggtggg ccagggcacg ggcagcttgc
      721 cggtggtgca gatcagcttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc
      781 cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca
      841 ccccggtgaa cagctcctcg cccttgctca ccatcggatc cttatcgtca tcgtcgtaca
      901 gatcacttcc accactattt gaacgcgcaa ccttttgact ggtagtctta aaatgctctg
      961 aagtggcctc taagtcttcg gttttattgc gcagatgctc aaggttctcg ccacgggcca
     1021 aaatacgctc tacattttgg gtcatgatat ttttcacgcc ttcgacctct gattgtaagt
     1081 tacgaacgcg gtcgttacca cctccacgac tacgcccacc tttatacagt tcatccatac
     1141 catgagtaat accagcggcg gtaacgaact ctaaaaggac catatggtcg cgtttctcat
     1201 tggggtcctt agacagggct gactgtgtgc tcaaatagtg attatcaggt aaaaggacgg
     1261 ggccatctcc aattggtgtg ttttgctggt aatggtctgc aagctggacg cttccatcct
     1321 caatgttgtg acgaatcttg aaatttgcct tgattccgtt tttttgttta tcagccgtga
     1381 tgtagacgtt atgagagata tagttgtatt ctaatttatg tcctaagatg ttgccatcct
     1441 ctttaaagtc gatacccttc aactcaatgc gattaacaag agtatctccc tcaaacttaa
     1501 cctcggcgcg ggtcttatag tttccatcat ctttaaaaaa aatggtgcgt tcctggacgt
     1561 aaccttctgg catcgcggac ttgaaaaagt cgtgttgttt catatggtca gggtagcgag
     1621 cgaaacactg cacgccccag gcgaacgtag taaccaatgt aggccacggt actggaagct
     1681 tgccggtagt gcaaataaat tttaatgtca actttccgta cgtcgcatcg ccctctcctt
     1741 ccccagatac ggaaaacttg tgaccattca catcgccatc cagttctaca agaatgggga
     1801 ccacgccggt aaacaattcc tcccccttag acatcaagtc ttcctcagag ataagttttt
     1861 gttccatgta tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat
     1921 ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct
     1981 ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta
     2041 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg
     2101 tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt
     2161 gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt
     2221 cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca
     2281 aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg
     2341 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt
     2401 cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg
     2461 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt
     2521 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg
     2581 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac
     2641 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg
     2701 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca
     2761 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
     2821 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
     2881 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
     2941 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
     3001 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
     3061 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
     3121 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
     3181 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
     3241 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
     3301 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
     3361 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     3421 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
     3481 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
     3541 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
     3601 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
     3661 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
     3721 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
     3781 gtgctcctgt cgttgaggac tcggctaggc tggcggggtt gccttactgg ttagcagaat
     3841 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacttc tgcgacctga
     3901 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     3961 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     4021 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     4081 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     4141 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     4201 aacagaaatc ccccttacac gtaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     4261 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     4321 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     4381 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     4441 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     4501 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     4561 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     4621 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     4681 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4741 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4801 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4861 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4921 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4981 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5041 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5101 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5161 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5221 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5281 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5341 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5401 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5461 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     5521 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     5581 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     5641 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     5701 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     5761 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     5821 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     5881 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     5941 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     6001 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     6061 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     6121 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     6181 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     6241 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     6301 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     6361 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6421 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     6481 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     6541 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     6601 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     6661 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     6721 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     6781 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     6841 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     6901 atgcgccgct acagggcgcg tcccattcgc ca
//
