LOCUS       sequence-456126-        5306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GEC-acceptor for 4G-cloning, designed for expression in E. coli 
            (Kanamyicin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5306)
  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 5306)
  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_K_ccdB
FEATURES             Location/Qualifiers
     source          1..5306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(590..1678)
                     /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     1640..1659
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1679..1756)
                     /gene="lacI"
                     /label=lacI promoter
     RBS             2041..2049
                     /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             2051..2359
                     /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     2238..2257
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2484..2503)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2694..2715
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2944..3759)
                     /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(2944..3759)
                     /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     3667..3686
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3751..3770
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3881..4469
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4370..4389
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4655..4794
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4720..4739)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4880..4902
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4896..5087)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
       61 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
      121 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
      181 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
      241 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
      301 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
      361 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
      421 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
      481 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
      541 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
      601 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
      661 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
      721 gctgattggc gttgccacct ccagtctggc cctgagagag ttgcagcaag cggtccacgc
      781 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
      841 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
      901 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
      961 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     1021 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     1081 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     1141 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     1201 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     1261 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     1321 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     1381 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     1441 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     1501 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     1561 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     1621 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     1681 tcaccaccct gaattgactc tcttccgggc gctatcatac cataccgcga aaggttttgc
     1741 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     1801 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     1861 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     1921 gcgctcatga gcccgaagct cttcccgatc ttaacgctct atggtctaaa gatttaaatg
     1981 gaattcctgc agcccaaact gcagttgaca acataaaaac tttgtgttat acttgtaacg
     2041 taaggaggta atgattcagt ttaaggttta cacctataaa agagagagcc gttatcgtct
     2101 gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct
     2161 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat
     2221 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat
     2281 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct
     2341 gatgttctgg ggaatataac gtctcaccat ttaaatctat atctcaatcg gggtttggga
     2401 ccggaagagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2461 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2521 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2581 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2641 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2701 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2761 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2821 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2881 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     2941 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     3001 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     3061 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     3121 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     3181 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     3241 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     3301 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     3361 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     3421 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     3481 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     3541 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     3601 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     3661 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     3721 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     3781 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3841 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3901 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3961 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4021 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4081 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4141 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4201 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4261 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4321 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4381 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4441 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4501 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4561 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4621 gcagcgagtc agtgagagag gaagcggatg agcgcctgat gcggtatttt ctccttacgc
     4681 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     4741 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4801 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4861 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4921 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     4981 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5041 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5101 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5161 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5221 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     5281 aatacagatg taggtgttcc acaggg
//
