LOCUS       sequence-454892-        3781 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GEC-acceptor for 4G-cloning, designed for expression in E. coli 
            (Streptomycin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3781)
  AUTHORS   Taschner M, Dickinson JB, Roisne-Hamelin F, Gruber S
  TITLE     4G cloning: rapid gene assembly for expression of multisubunit 
            protein complexes in diverse hosts
  JOURNAL   bioRxiv 2024.06.17.599261
REFERENCE   2  (bases 1 to 3781)
  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"; journalName: "bioRxiv 
            2024.06.17.599261"
COMMENT     Sequence Label: pMulti_S_ccdB
FEATURES             Location/Qualifiers
     source          1..3781
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(59..1141)
                     /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     1103..1122
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1142..1219)
                     /gene="lacI"
                     /label=lacI promoter
     RBS             1368..1376
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1378..1686
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MIQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSD
                     KVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1565..1584
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1896..2687)
                     /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"
     promoter        complement(2688..2779)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
       61 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      121 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
      181 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
      241 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
      301 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
      361 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
      421 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
      481 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
      541 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
      601 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      661 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      721 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      781 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      841 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      901 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      961 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     1021 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     1081 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     1141 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     1201 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     1261 aaatgctctt cccgatctta acgctctatg gtctaaagat ttaaatggaa ttcctgcagc
     1321 ccaaactgca gttgacaaca taaaaacttt gtgttatact tgtaacgtaa ggaggtaatg
     1381 attcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta
     1441 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt
     1501 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc
     1561 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg
     1621 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
     1681 atataacgtc tcaccattta aatctatatc tcaatcgggg tttgggaccg gaagagcgca
     1741 tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag
     1801 acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcatc gtggccggat
     1861 cttgcggccc ctcggcttga acgaattgtt agacattatt tgccgactac cttggtgatc
     1921 tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc caagcgatct
     1981 tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata ctgggccggc
     2041 aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg
     2101 ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc
     2161 ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc
     2221 ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt
     2281 gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg
     2341 tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg
     2401 atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg
     2461 gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt
     2521 acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg
     2581 gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact
     2641 acctctgata gttgagtcga tacttcggcg atcaccgctt ccctcatact cttccttttt
     2701 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     2761 atttagaaaa ataaacaaat agctagctca ctcggtcgct acgctccggg cgtgagactg
     2821 cggcgggcgc tgcggacaca tacaaagtta cccacagatt ccgtggataa gcaggggact
     2881 aacatgtgag gcaaaacagc agggccgcgc cggtggcgtt tttccatagg ctccgccctc
     2941 ctgccagagt tcacataaac agacgctttt ccggtgcatc tgtgggagcc gtgaggctca
     3001 accatgaatc tgacagtacg ggcgaaaccc gacaggactt aaagatcccc accgtttccg
     3061 gcgggtcgct ccctcttgcg ctctcctgtt ccgaccctgc cgtttaccgg atacctgttc
     3121 cgcctttctc ccttacggga agtgtggcgc tttctcatag ctcacacact ggtatctcgg
     3181 ctcggtgtag gtcgttcgct ccaagctggg ctgtaagcaa gaactccccg ttcagcccga
     3241 ctgctgcgcc ttatccggta actgttcact tgagtccaac ccggaaaagc acggtaaaac
     3301 gccactggca gcagccattg gtaactggga gttcgcagag gatttgttta gctaaacacg
     3361 cggttgctct tgaagtgtgc gccaaagtcc ggctacactg gaaggacaga tttggttgct
     3421 gtgctctgcg aaagccagtt accacggtta agcagttccc caactgactt aaccttcgat
     3481 caaaccacct ccccaggtgg ttttttcgaa ggttaagtca gttggggaac tgcttaacct
     3541 tcgatcaaac cacctcccca ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga
     3601 aaaaaaggat ctcaagaaga tcctttgatc ttttctactg aaccgctcta gatttcagtg
     3661 caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc
     3721 tcatgttagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     3781 g
//
