LOCUS       sequence-310687-        5829 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses calpain-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 5829)
  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 5829)
  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-Calpain
FEATURES             Location/Qualifiers
     source          1..5829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..59
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     143..162
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        143..161
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    162..186
                     /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             201..223
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             836..853
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(906..924)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      920..967
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1004..1459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1091..1110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1301..1322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1551..2366)
                     /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(1551..2366)
                     /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     2274..2293
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2358..2377
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2488..3076
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2977..2996
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3230..3247
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3262..3401
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3327..3346)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3487..3509
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3503..3694)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4503..5585)
                     /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     5547..5566
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5586..5663)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
       61 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
      121 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
      181 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tggggatatc
      241 tctgatggca aaaattcgtc aagaacatca gagcattgca gaaaaagttg cagaactgag
      301 cctggaaacc cgtgaatttc tgagcgttac caccgcaaaa cgccaagaac aggcagaaaa
      361 acaggcacaa gaactgcagg cattctataa agatctgcaa gaaaccagcc agggcagtgg
      421 ttcgggacaa caagaagtat atggcatgat gccccgcgac ggctcgggta gcggtcaagc
      481 ccaagagctg ctggcctttc acaaagaact gcaagagact tctcagcagt tcctgtcagc
      541 aacagcccaa gcccgtatcg cccaagcgga gaaacaggcg caagagctgc tggcatttta
      601 tcaagaagtt cgcgaaacct cacagcaatt tctgtcagcc actgcgcaag cacgcattgc
      661 gcaagctgaa aaacaagcgc aagagctgct ggcgttccat aaagagctgc aagaaacgtc
      721 acaacagttt ctgagcgcaa cggccgatgc acgtacagca caggcgaaag aacagaaaga
      781 aagcctgctg aaatttcgtc aggacctgtt tgttagcatt ttcggcagcc tcgagcacca
      841 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
      901 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
      961 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     1021 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1081 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1141 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1201 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1261 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1321 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1381 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     1441 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1501 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     1561 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     1621 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     1681 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     1741 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     1801 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     1861 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     1921 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     1981 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     2041 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     2101 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     2161 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     2221 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     2281 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     2341 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     2401 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2461 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2521 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2581 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2641 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2701 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2761 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2821 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2881 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2941 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3001 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3061 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3121 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3181 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3241 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3301 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3361 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3421 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3481 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3541 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3601 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3661 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     3721 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     3781 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     3841 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     3901 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     3961 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4021 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4081 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4141 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4201 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4261 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4321 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4381 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4441 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4501 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4561 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4621 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     4681 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     4741 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     4801 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     4861 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     4921 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     4981 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5041 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5101 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5161 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5221 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5281 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5341 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5401 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5461 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5521 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5581 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     5641 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     5701 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     5761 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     5821 gaaacaagc
//
