LOCUS       sequence-329574-        6929 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses eCFP-Sec22b(A131-H190)-mVenus in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6929)
  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 6929)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Sec22b
FEATURES             Location/Qualifiers
     source          1..6929
                     /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
                     ANFKIRHNIEDGGVQLADHYQQNNPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     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(1832..1861)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     RBS             1870..1892
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1907..1931
                     /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(1931..1950)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1932..1950)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2034..2053)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2259..2336
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2337..3419
                     /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(2356..2375)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4228..4419
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4413..4435)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4521..4660
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4576..4595
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4675..4692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4846..5434)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4926..4945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5545..5564)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5556..6371
                     /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             5556..6371
                     /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(5629..5648)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6463..6918)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6600..6621)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6812..6831
                     /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 ggggttgttc 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 gagtgcatat tcaggtattt cgcatcctgg cgatacttct
      961 ttgacaggga ggacaggtta ttggctttag aatctaaggc ggaaagcgcc tctccacgtt
     1021 gaaggacttc ttcgatgttg gcgaccataa tacgctgcac atcttgtaat tccgtattga
     1081 ttgagcctaa gttacgacgc gcacgactac 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 tcaagtcttc ctcagagata agtttttgtt
     1861 ccatgtatat ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg
     1921 ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta
     1981 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
     2041 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
     2101 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
     2161 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     2221 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
     2281 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     2341 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     2401 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     2461 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     2521 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     2581 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     2641 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     2701 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     2761 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     2821 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     2881 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     2941 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     3001 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     3061 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     3121 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     3181 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     3241 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     3301 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     3361 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     3421 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
     3481 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     3541 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg
     3601 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg
     3661 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt
     3721 ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg
     3781 ctcctgtcgt tgaggactcg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     3841 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacttctgc gacctgagca
     3901 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     3961 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     4021 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     4081 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     4141 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     4201 agaaatcccc cttacacgta ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     4261 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     4321 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc
     4381 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4441 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     4501 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     4561 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga
     4621 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     4681 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4741 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4801 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4861 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4921 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4981 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5041 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5101 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5161 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5221 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5281 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5341 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5401 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5461 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac
     5521 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     5581 cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     5641 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg
     5701 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     5761 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     5821 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg
     5881 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     5941 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     6001 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     6061 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     6121 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     6181 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     6241 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     6301 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     6361 agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa
     6421 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     6481 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     6541 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     6601 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     6661 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     6721 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     6781 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     6841 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     6901 cgccgctaca gggcgcgtcc cattcgcca
//
