LOCUS       sequence-310690-        5824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TEV-sensitive variant of Anabaena flos-aquae GvpC in 
            E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5824)
  AUTHORS   Lakshmanan A, Jin Z, Nety SP, Sawyer DP, Lee-Gosselin A, Malounda D,
            Swift MB, Maresca D, Shapiro MG
  TITLE     Acoustic biosensors for ultrasound imaging of enzyme activity.
  JOURNAL   Nat Chem Biol. 2020 Sep;16(9):988-996. doi: 
            10.1038/s41589-020-0591-0. Epub 2020 Jul 13.
  PUBMED    32661379
REFERENCE   2  (bases 1 to 5824)
  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: 
            "10.1038/s41589-020-0591-0"; journalName: "Nat Chem Biol"; date: 
            "2020-09"; volume: "16"; issue: "9"; pages: "988-996"
COMMENT     Sequence Label: C-His-GvpC-TEV
FEATURES             Location/Qualifiers
     source          1..5824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     564..583
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(603..680)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     886..905
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     989..1008
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        989..1007
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1008..1032
                     /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)."
     RBS             1047..1069
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1299..1319
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1677..1694
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1747..1765)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1761..1808
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1845..2300
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1932..1951)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2142..2163
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2392..3207)
                     /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             complement(2392..3207)
                     /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     3115..3134
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3199..3218
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3329..3917
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3818..3837
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4071..4088
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4103..4242
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4168..4187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4328..4350
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4344..4535)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5344..5824,1..602))
                     /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"
ORIGIN
        1 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
       61 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
      121 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
      181 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
      241 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
      301 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
      361 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
      421 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
      481 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
      541 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
      601 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
      661 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
      721 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
      781 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
      841 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      901 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      961 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     1021 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatga
     1081 tctcattaat ggctaaaatt cgccaggaac atcagagcat tgccgaaaaa gtcgcggagt
     1141 tgagcttgga aacgcgcgag tttctgtccg ttaccactgc gaaacgccag gagcaggctg
     1201 aaaaacaagc ccaggagctg caggcattct acaaagattt gcaggaaacg tcccagcagt
     1261 tcctgtccga gactgcgggt tctggttctg gttctggtga gaatctgtat tttcaaggaa
     1321 gcggaagcgg aagcggattt cataaggaac tgcaggaaac ctcacaacag ttcttatcag
     1381 caaccgcaca ggcgcgtatt gcgcaggctg aaaaacaagc gcaagaattg ctcgcgttct
     1441 atcaggaggt ccgcgagacg agccagcagt ttttaagcgc gacagcgcag gcccgcattg
     1501 cgcaggcgga gaagcaggcc caagagctcc tggccttcca taaagaactt caagaaacgt
     1561 cccaacagtt cttaagcgca accgcggacg ctcgtacggc gcaggccaag gagcagaaag
     1621 agagcctgct caagttccgt caggatctgt ttgtgtctat ttttggcagc ctcgagcatc
     1681 accaccatca tcactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1741 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1801 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1861 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1921 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1981 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2041 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2101 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2161 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2221 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2281 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2341 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     2401 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     2461 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     2521 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     2581 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2641 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2701 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2761 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2821 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2881 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2941 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     3001 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     3061 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     3121 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     3181 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     3241 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3301 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3361 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3421 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     3481 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3541 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3601 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3661 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3721 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3781 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3841 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3901 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3961 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4021 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4081 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4141 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4201 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     4261 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4321 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     4381 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     4441 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     4501 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     4561 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     4621 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     4681 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4741 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4801 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4861 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4921 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4981 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     5041 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     5101 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     5161 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     5221 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     5281 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     5341 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5401 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     5461 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     5521 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5581 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     5641 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5701 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5761 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5821 atgc
//
