LOCUS       sequence-444927-        6332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FRB-Linker-Large BiT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6332)
  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 6332)
  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)-FRB-Lg
FEATURES             Location/Qualifiers
     source          1..6332
                     /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     2430..2447
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2462..2601
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2527..2546)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2687..2709
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2703..2894)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3703..4785)
                     /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     4747..4766
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4786..4863)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5069..5088
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5172..5191
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5172..5190
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5191..5215
                     /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             5230..5252
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5272..5289
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5293..5325
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(5329..5337,5338..5352)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(6247..6265)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6261..6308
                     /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 gaaaaaacca cctggggagg tggtttgatc
     1561 gaaggttaag cagttcccca actgacttaa ccttcgaaaa aaccacctgg ggaggtggtt
     1621 tgatcgaagg ttaagtcagt tggggaactg cttaaccgtg gtaactggct ttcgcagagc
     1681 acagcaacca aatctgtcct tccagtgtag ccggactttg gcgcacactt caagagcaac
     1741 cgcgtgttta gctaaacaaa tcctctgcga actcccagtt accaatggct gctgccagtg
     1801 gcgttttacc gtgcttttcc gggttggact caagtgaaca gttaccggat aaggcgcagc
     1861 agtcgggctg aacggggagt tcttgcttac agcccagctt ggagcgaacg acctacaccg
     1921 agccgagata ccagtgtgtg agctatgaga aagcgccaca cttcccgtaa gggagaaagg
     1981 cggaacaggt atccggtaaa cggcagggtc ggaacaggag agcgcaagag ggagcgaccc
     2041 gccggaaacg gtggggatct ttaagtcctg tcgggtttcg cccgtactgt cagattcatg
     2101 gttgagcctc acggctccca cagatgcacc ggaaaagcgt ctgtttatgt gaactctggc
     2161 aggagggcgg agcctatgga aaaacgccac cggcgcggcc ctgctgtttt gcctcacatg
     2221 ttagtcccct gcttatccac ggaatctgtg ggtaactttg tatgtgtccg cagcgcaacg
     2281 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2341 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2401 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2461 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     2521 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     2581 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2641 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     2701 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     2761 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     2821 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     2881 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     2941 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3001 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     3061 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     3121 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     3181 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     3241 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     3301 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     3361 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     3421 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     3481 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     3541 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     3601 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     3661 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     3721 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3781 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     3841 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     3901 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     3961 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4021 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     4081 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     4141 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     4201 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     4261 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     4321 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     4381 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     4441 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     4501 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     4561 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     4621 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     4681 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     4741 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     4801 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     4861 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     4921 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     4981 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     5041 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     5101 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     5161 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     5221 agaaataatt ttgtttaact ttaagaagga gatatacata tggggggttc tcatcatcat
     5281 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
     5341 gatgacgata aggatccgag catcctgtgg catgaaatgt ggcatgaagg cctggaagaa
     5401 gcgagccgtc tgtattttgg cgaacgtaac gtgaaaggca tgtttgaagt gctggaaccg
     5461 ctgcatgcca tgatggaacg tggcccgcag accctgaaag aaaccagctt taaccaggcg
     5521 tatggccgtg atctgatgga agcgcaggaa tggtgccgta aatacatgaa aagcggcaac
     5581 gtgaaagatc tgctgcaagc gtgggatctg tattatcatg tgtttcgccg catcagcgtt
     5641 tctcaaggca gttcaggtgg tggcgggagc ggaggtggag gctcgagcgg tgtcttcaca
     5701 ctcgaagatt tcgttgggga ctgggaacag acagccgcct acaacctgga ccaagtcctt
     5761 gaacagggag gtgtgtccag tttgctgcag aatctcgccg tgtccgtaac tccgatccaa
     5821 aggattgtcc ggagcggtga aaatgccctg aagatcgaca tccatgtcat catcccgtat
     5881 gaaggtctga gcgccgacca aatggcccag atcgaagagg tgtttaaggt ggtgtaccct
     5941 gtggatgatc atcactttaa ggtgatcctg ccctatggca cactggtaat cgacggggtt
     6001 acgccgaaca tgctgaacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc
     6061 aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg
     6121 atcacccccg acggctccat gctgttccga gtaaccatca acagctgagg ctgttttggc
     6181 ggatgagaga agattgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     6241 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     6301 gttttttgct gaaaggagga actatatccg ga
//
