LOCUS       sequence-450699-        5439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GEC-acceptor for 4G-cloning, designed for expression in E. coli 
            (Ampicillin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5439)
  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 5439)
  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_A_ccdB
FEATURES             Location/Qualifiers
     source          1..5439
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     216..238
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(232..423)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1232..2320)
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQTGGGNANQLLPVSLVKRKTTLAPNTQTAS
                     PRALADSLMQLARQVSRLESGQ"
     primer_bind     2282..2301
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2321..2398)
                     /gene="lacI"
                     /label=lacI promoter
     RBS             2683..2691
                     /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             2693..3001
                     /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="MIQFKVYTYKRESRYRLFVDV*SDIIDTPGRRMVIPLASARLLSD
                     KVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2880..2899
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(3126..3145)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3336..3357
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3520..3624
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3625..3693,3694..4485)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3843..3862)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4656..5244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5145..5164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    join(5430..5439,1..130)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
      121 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
      181 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
      241 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
      301 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
      361 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
      421 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
      481 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
      541 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
      601 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
      661 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
      721 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
      781 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
      841 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
      901 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
      961 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     1021 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     1081 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     1141 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     1201 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1261 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1321 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg gcgttgccac
     1381 ctccagtctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     1441 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     1501 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     1561 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     1621 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     1681 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     1741 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     1801 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     1861 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     1921 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     1981 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     2041 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     2101 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     2161 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     2221 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     2281 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     2341 tctcttccgg gcgctatcat accataccgc gaaaggtttt gcgccattcg atggtgtccg
     2401 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     2461 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     2521 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     2581 ctcttcccga tcttaacgct ctatggtcta aagatttaaa tggaattcct gcagcccaaa
     2641 ctgcagttga caacataaaa actttgtgtt atacttgtaa cgtaaggagg taatgattca
     2701 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtatagag
     2761 tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     2821 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     2881 catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga
     2941 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
     3001 acgtctcacc atttaaatct atatctcaat cggggtttgg gaccggaaga gcggcgcatt
     3061 aaccgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3121 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3181 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3241 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3301 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3361 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3421 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3481 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3541 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3601 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     3661 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     3721 gatgctgaag atcagttggg tgcacgggtg ggttacatcg aactggatct caacagcggt
     3781 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     3841 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     3901 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     3961 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4021 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4081 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4141 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     4201 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4261 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4321 tctggagccg gtgagcgtgg gtcccgcggt atcattgcag cactggggcc agatggtaag
     4381 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4441 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4501 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4561 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4621 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4681 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4741 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4801 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4861 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4921 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4981 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5041 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5101 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5161 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5221 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5281 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5341 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     5401 gagtcagtga gagaggaagc ggatgagcgc ctgatgcgg
//
