LOCUS       sequence-445950-        2017 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for bacterial promoter and terminator, N-terminal 
            tag added.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2017)
  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 2017)
  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: pD(P+T)_T7N
FEATURES             Location/Qualifiers
     source          1..2017
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        174..276
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             277..936
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(343..362)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      1331..1687
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(1740..1773)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1840..1859
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1840..1858
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1859..1883
                     /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             1898..1920
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      1964..2011
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gagacccact gcttgagcct agaagatccg gctgctaaca aagcccgaaa ggaagctgag
       61 ttggctgctg tcaccgctga gcaataacta tcataacccc taggtgccat ttcattacct
      121 ctttctccgc acccgacata gatctgggcc aacttttggc gaaaatgaga cgttgatcgg
      181 cacgtaagag ggtccaactt tcaccataat gaaataagat cactaccggg cgtatttttt
      241 gagttatcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat
      301 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
      361 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
      421 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
      481 gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
      541 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa
      601 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt
      661 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat
      721 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc
      781 cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
      841 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
      901 caacagtact gcgatgagtg gcagggtggg gcgtaatttt tttaaggcag ttattggtgc
      961 ccttaaacgc ctggttgcta cgcctgaata agtgataata agcggatgaa tggcagaaat
     1021 tcgaaagcaa attcgacccg gtcgtcggtt cagggcaggg tcgttaaata gccgcttatg
     1081 tctattgctg gtttaccggt ttattgacta ccggaagcag tgtgaccgtg tgcttctcaa
     1141 atgcctgagg ccagtttgct caggctctcc ccgtggaggt aataattgac gatatgatca
     1201 tttattctgc ctcccagctg acattcatcc ggggtcagca ccgtttctgc ggactggctt
     1261 tctacgtgtt ccgcttcctt tagcagccct tgcgccctga gtgcttgcgg cagcgtgaag
     1321 ctaattccca tgtcagccgt taagtgttcc tgtgtcactc aaaattgctt tgagaggctc
     1381 taagggcttc tcagtgcgtt acatccctgg cttgttgtcc acaaccgtta aaccttaaaa
     1441 gctttaaaag ccttatatat tctttttttt cttataaaac ttaaaacctt agaggctatt
     1501 taagttgctg atttatatta attttattgt tcaaacatga gagcttagta cgtgaaacat
     1561 gagagcttag tacgttagcc atgagagctt agtacgttag ccatgagggt ttagttcgtt
     1621 aaacatgaga gcttagtacg ttaaacatga gagcttagta cgtgaaacat gagagcttag
     1681 tacgtactat caacaggttg aactgctgat caacagatcc tctacgcggc cgcggtacca
     1741 taacttcgta tagcatacat tatacgaagt tatctgccag gcacatgggt tttactagta
     1801 tcgattcgcg acctactccg gaatattagg tctcagcgat aatacgactc actatagggg
     1861 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
     1921 tataaccatg gcgagagacc acgtgcaact ggtctccgga taactagcat aaccccttgg
     1981 ggcctctaaa cgggtcttga ggggtttttt ggtacgg
//
