LOCUS       sequence-445069-        6256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Large BiT-Linker-GvpK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6256)
  AUTHORS   Iburg M, Anderson AP, Wong VT, Anton ED, He A, Lu GJ
  TITLE     Elucidating the assembly of gas vesicles by systematic 
            protein-protein interaction analysis.
  JOURNAL   EMBO J. 2024 Oct;43(19):4156-4172. doi: 10.1038/s44318-024-00178-2. 
            Epub 2024 Sep 3.
  PUBMED    39227754
REFERENCE   2  (bases 1 to 6256)
  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/s44318-024-00178-2"; journalName: "EMBO J"; date: 
            "2024-10"; volume: "43"; issue: "19"; pages: "4156-4172"
COMMENT     Sequence Label: pET26b(modified)-Lg-K
FEATURES             Location/Qualifiers
     source          1..6256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      13..468
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(100..119)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     310..331
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(560..1351)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     primer_bind     2429..2446
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2461..2600
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2526..2545)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2686..2708
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2702..2893)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3702..4784)
                     /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     4746..4765
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4785..4862)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5068..5087
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5171..5190
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5171..5189
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5190..5214
                     /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             5229..5251
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5262..5285
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(6171..6189)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6185..6232
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
       61 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      121 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      181 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      241 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      301 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
      361 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
      421 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tatttgccga ctaccttggt gatctcgcct ttcacgtagt
      601 ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat
      661 aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc
      721 agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg
      781 acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag ttccatagcg
      841 ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct
      901 ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca
      961 gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt
     1021 ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa
     1081 caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca
     1141 aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc accgtaacca
     1201 gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg tacaaatgta
     1261 cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag
     1321 tcgatacttc ggcgatcacc gcttccctca taacacccct tgtattactg tttatgtaag
     1381 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     1441 tcagaccccg tagaaaagat caaaggatct tcgatcaaag gatcttcttg agatcctttt
     1501 tttctgcgcg taatcttttg ccctgtaaac gaaaaaccac ctggggaggt ggtttgatcg
     1561 aaggttaagc agttccccaa ctgacttaac cttcgaaaaa accacctggg gaggtggttt
     1621 gatcgaaggt taagtcagtt ggggaactgc ttaaccgtgg taactggctt tcgcagagca
     1681 cagcaaccaa atctgtcctt ccagtgtagc cggactttgg cgcacacttc aagagcaacc
     1741 gcgtgtttag ctaaacaaat cctctgcgaa ctcccagtta ccaatggctg ctgccagtgg
     1801 cgttttaccg tgcttttccg ggttggactc aagtgaacag ttaccggata aggcgcagca
     1861 gtcgggctga acggggagtt cttgcttaca gcccagcttg gagcgaacga cctacaccga
     1921 gccgagatac cagtgtgtga gctatgagaa agcgccacac ttcccgtaag ggagaaaggc
     1981 ggaacaggta tccggtaaac ggcagggtcg gaacaggaga gcgcaagagg gagcgacccg
     2041 ccggaaacgg tggggatctt taagtcctgt cgggtttcgc ccgtactgtc agattcatgg
     2101 ttgagcctca cggctcccac agatgcaccg gaaaagcgtc tgtttatgtg aactctggca
     2161 ggagggcgga gcctatggaa aaacgccacc ggcgcggccc tgctgttttg cctcacatgt
     2221 tagtcccctg cttatccacg gaatctgtgg gtaactttgt atgtgtccgc agcgcaacgc
     2281 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     2341 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     2401 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     2461 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     2521 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     2581 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2641 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2701 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     2761 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     2821 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     2881 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     2941 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     3001 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     3061 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     3121 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     3181 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     3241 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     3301 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     3361 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     3421 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     3481 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     3541 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     3601 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     3661 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     3721 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3781 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     3841 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     3901 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     3961 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     4021 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     4081 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     4141 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     4201 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     4261 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     4321 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     4381 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     4441 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     4501 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     4561 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     4621 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     4681 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     4741 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     4801 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     4861 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     4921 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     4981 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     5041 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     5101 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     5161 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     5221 gaaataattt tgtttaactt taagaaggag atatacatat ggattataaa gatgatgatg
     5281 ataaagtctt cacactcgaa gatttcgttg gggactggga acagacagcc gcctacaacc
     5341 tggaccaagt ccttgaacag ggaggtgtgt ccagtttgct gcagaatctc gccgtgtccg
     5401 taactccgat ccaaaggatt gtccggagcg gtgaaaatgc cctgaagatc gacatccatg
     5461 tcatcatccc gtatgaaggt ctgagcgccg accaaatggc ccagatcgaa gaggtgttta
     5521 aggtggtgta ccctgtggat gatcatcact ttaaggtgat cctgccctat ggcacactgg
     5581 taatcgacgg ggttacgccg aacatgctga actatttcgg acggccgtat gaaggcatcg
     5641 ccgtgttcga cggcaaaaag atcactgtaa cagggaccct gtggaacggc aacaaaatta
     5701 tcgacgagcg cctgatcacc cccgacggct ccatgctgtt ccgagtaacc atcaacgttt
     5761 ctcaaggcag ttcaggtggt ggcgggagcg gaggtggagg ctcgagcggt caaccggtca
     5821 gccaagcaaa tggacgaatc cacttggatc ctgatcaagc tgaacaaggc ttagcgcagc
     5881 ttgtgatgac agttattgag ctattgaggc aaatcgttga acgtcatgcc atgaggcggg
     5941 tggagggtgg aacgttgacg gacgaacaaa ttgaaaactt aggaattgca ctaatgaact
     6001 tagaagaaaa aatggacgag ttgaaagagg tgttcggtct ggatgcagaa gatttaaata
     6061 ttgatcttgg accgctaggc agcctgcttt aaggctgttt tggcggatga gagaagattg
     6121 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     6181 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     6241 aggaactata tccgga
//
