LOCUS       sequence-329622-        6896 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eCFP-VAMP4(P48-R107)-mVenus in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6896)
  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 6896)
  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-CV-VAMP4
FEATURES             Location/Qualifiers
     source          1..6896
                     /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"
     RBS             1837..1859
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1874..1898
                     /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(1898..1917)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1899..1917)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2001..2020)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2226..2303
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2304..3386
                     /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(2323..2342)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4195..4386
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4380..4402)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4488..4627
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4543..4562
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4642..4659)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4813..5401)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4893..4912)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5512..5531)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5523..6338
                     /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             5523..6338
                     /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(5596..5615)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6430..6885)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6567..6588)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6779..6798
                     /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 gagcggcgca gttgcttact acgattagag aaggcggtgg
      961 cattgtctga aagagattca ctcttgtcct gtaattcatc aaggcgctct ccacgttcaa
     1021 ttaccttagt aatgttctcc tgcattacgt caatcacttc gtctacttga ttttggacgt
     1081 gtttgatttt gtcgttacgt ggacgactac gcccaccttt atacagttca tccataccat
     1141 gagtaatacc agcggcggta acgaactcta aaaggaccat atggtcgcgt ttctcattgg
     1201 ggtccttaga cagggctgac tgtgtgctca aatagtgatt atcaggtaaa aggacggggc
     1261 catctccaat tggtgtgttt tgctggtaat ggtctgcaag ctggacgctt ccatcctcaa
     1321 tgttgtgacg aatcttgaaa tttgccttga ttccgttttt ttgtttatca gccgtgatgt
     1381 agacgttatg agagatatag ttgtattcta atttatgtcc taagatgttg ccatcctctt
     1441 taaagtcgat acccttcaac tcaatgcgat taacaagagt atctccctca aacttaacct
     1501 cggcgcgggt cttatagttt ccatcatctt taaaaaaaat ggtgcgttcc tggacgtaac
     1561 cttctggcat cgcggacttg aaaaagtcgt gttgtttcat atggtcaggg tagcgagcga
     1621 aacactgcac gccccaggcg aacgtagtaa ccaatgtagg ccacggtact ggaagcttgc
     1681 cggtagtgca aataaatttt aatgtcaact ttccgtacgt cgcatcgccc tctccttccc
     1741 cagatacgga aaacttgtga ccattcacat cgccatccag ttctacaaga atggggacca
     1801 cgccggtaaa caattcctcc cccttagaca tgtatatctc cttcttaaag ttaaacaaaa
     1861 ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt
     1921 tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
     1981 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
     2041 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg
     2101 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg
     2161 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga
     2221 tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     2281 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     2341 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     2401 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     2461 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     2521 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     2581 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     2641 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     2701 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     2761 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     2821 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     2881 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     2941 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     3001 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     3061 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     3121 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     3181 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     3241 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     3301 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     3361 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     3421 taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     3481 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc
     3541 gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc
     3601 gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc
     3661 ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg
     3721 gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggactcggct aggctggcgg
     3781 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc
     3841 tgctgcaaaa cttctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg
     3901 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca
     3961 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga
     4021 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa
     4081 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
     4141 ttcatcggta tcattacccc catgaacaga aatccccctt acacgtaggc atcagtgacc
     4201 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     4261 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac
     4321 gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     4381 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     4441 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     4501 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     4561 tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     4621 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4681 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4741 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4801 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4861 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4921 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4981 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5041 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5101 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5161 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5221 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     5281 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5341 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5401 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5461 agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
     5521 ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg
     5581 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     5641 tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     5701 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     5761 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     5821 cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     5881 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     5941 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     6001 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     6061 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     6121 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     6181 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     6241 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     6301 aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat gagcggatac
     6361 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6421 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     6481 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     6541 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     6601 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     6661 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     6721 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     6781 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     6841 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
