LOCUS       sequence-329551-        6914 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eCFP-SNAP25(A141-G206)-mVenus in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6914)
  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 6914)
  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-SNAP25
FEATURES             Location/Qualifiers
     source          1..6914
                     /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             1855..1877
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1892..1916
                     /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(1916..1935)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1917..1935)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2019..2038)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2244..2321
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2322..3404
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSKGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2341..2360)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4213..4404
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4398..4420)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4506..4645
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4561..4580
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4660..4677)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4831..5419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4911..4930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5530..5549)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5541..6356
                     /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             5541..6356
                     /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(5614..5633)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6448..6903)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6585..6606)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6797..6816
                     /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 gacccagacc ccagcatttt ggtcgcacgt tggttagcct
      961 cgtcaatgcg tgtcttattg ctatcagcct tttccatgat gcgatcaatc tgacgatttt
     1021 gggtatcaat ttcattgccc atatctaatg ccatatgacg caagttgcca atgatcccag
     1081 atacctgctc aaggttctca tccatttcat tttcgcgggc acgactacgc ccacctttat
     1141 acagttcatc cataccatga gtaataccag cggcggtaac gaactctaaa aggaccatat
     1201 ggtcgcgttt ctcattgggg tccttagaca gggctgactg tgtgctcaaa tagtgattat
     1261 caggtaaaag gacggggcca tctccaattg gtgtgttttg ctggtaatgg tctgcaagct
     1321 ggacgcttcc atcctcaatg ttgtgacgaa tcttgaaatt tgccttgatt ccgttttttt
     1381 gtttatcagc cgtgatgtag acgttatgag agatatagtt gtattctaat ttatgtccta
     1441 agatgttgcc atcctcttta aagtcgatac ccttcaactc aatgcgatta acaagagtat
     1501 ctccctcaaa cttaacctcg gcgcgggtct tatagtttcc atcatcttta aaaaaaatgg
     1561 tgcgttcctg gacgtaacct tctggcatcg cggacttgaa aaagtcgtgt tgtttcatat
     1621 ggtcagggta gcgagcgaaa cactgcacgc cccaggcgaa cgtagtaacc aatgtaggcc
     1681 acggtactgg aagcttgccg gtagtgcaaa taaattttaa tgtcaacttt ccgtacgtcg
     1741 catcgccctc tccttcccca gatacggaaa acttgtgacc attcacatcg ccatccagtt
     1801 ctacaagaat ggggaccacg ccggtaaaca attcctcccc cttagacatg tatatctcct
     1861 tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta
     1921 tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt
     1981 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
     2041 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
     2101 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
     2161 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     2221 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
     2281 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     2341 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     2401 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     2461 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     2521 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     2581 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     2641 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     2701 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     2761 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     2821 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     2881 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     2941 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     3001 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     3061 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     3121 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     3181 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     3241 tctctaaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     3301 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     3361 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     3421 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     3481 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     3541 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag
     3601 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg
     3661 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag
     3721 gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg
     3781 actcggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag
     3841 cgaacgtgaa gcgactgctg ctgcaaaact tctgcgacct gagcaacaac atgaatggtc
     3901 ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg
     3961 ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta
     4021 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc
     4081 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc
     4141 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac
     4201 acgtaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga
     4261 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac
     4321 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc
     4381 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4441 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4501 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     4561 cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg
     4621 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4681 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4741 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4801 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4861 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4921 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4981 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5041 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5101 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5161 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5221 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     5281 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5341 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5401 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5461 gaacgaaaac tcacgttaag ggattttggt catgaacaat aaaactgtct gcttacataa
     5521 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc
     5581 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     5641 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc
     5701 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     5761 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     5821 catggttact caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc
     5881 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     5941 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     6001 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     6061 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     6121 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     6181 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     6241 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     6301 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat
     6361 taattcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6421 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     6481 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     6541 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     6601 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     6661 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     6721 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     6781 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     6841 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     6901 cgtcccattc gcca
//
