LOCUS       sequence-151922-        4104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E coli genetic modification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4104)
  AUTHORS   Yang J, Sun B, Huang H, Jiang Y, Diao L, Chen B, Xu C, Wang X, Liu 
            J, Jiang W, Yang S
  TITLE     High-Efficiency Scarless Genetic Modification in Escherichia coli by
            Using Lambda Red Recombination and I-SceI Cleavage.
  JOURNAL   Appl Environ Microbiol. 2014 Jul 1;80(13):3826-3834. Epub 2014 Apr 
            18.
  PUBMED    24747889
REFERENCE   2  (bases 1 to 4104)
  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: "Appl 
            Environ Microbiol."; date: "2014-07-1"; volume: "80(13)"; pages: 
            "3826-3834. Epub 2014 Apr 18"
COMMENT     Sequence Label: pMDIAI
FEATURES             Location/Qualifiers
     source          1..4104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..27)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10..26)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(19..41)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(235..254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     354..376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(414..432)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        500..604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(605..673,674..1465)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(823..842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1636..2224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2125..2144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2378..2395
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2512..2533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2548..2553,2554..2571,2572..2578)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2586..2602
                     /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)."
     primer_bind     2591..2613
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2610..2626
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2610..2626
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2721..2754
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     oriT            2761..2870
                     /note="incP origin of transfer"
     CDS             3186..3986
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     protein_bind    3996..4042
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 ttctttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
       61 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
      121 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
      181 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
      241 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
      301 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
      361 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
      421 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
      481 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
      541 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      601 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
      661 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
      721 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
      781 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
      841 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
      901 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
      961 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1021 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1081 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1141 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     1201 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     1261 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     1321 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     1381 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     1441 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1501 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1561 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1621 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1681 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1741 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1801 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1861 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1921 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1981 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2041 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2101 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2161 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2221 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2281 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2341 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2401 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2461 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     2521 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     2581 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgaact
     2641 cgtcctcggt tcccggcgat cctctggaga ttatattacc ctgttatccc taattccggg
     2701 gatccgtcga cctgcagttc gaagttccta ttctctagaa agtataggaa cttcgaagtt
     2761 cccgccagcc tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag
     2821 tgaagaagga acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc
     2881 gttggataca ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga
     2941 aaaaggatgg atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg
     3001 aaaatgcagc tcacggtaac tgatgccgta tttgcagtac cagcgtacgg cccacagaat
     3061 gatgtcacgc tgaaaatgcc ggcctttgaa tgggttcatg tgcagctcca taagcaaaag
     3121 gggatgataa gtttatcacc accgactatt tgcaacagtg ccgttgatcg tgctatgatc
     3181 gactgatgtc atcagcggtg gagtgcaatg tcgtgcaata cgaatggcga aaagccgagc
     3241 tcatcggtca gcttctcaac cttggggtta cccccggcgg tgtgctgctg gtccacagct
     3301 ccttccgtag cgtccggccc ctcgaagatg ggccacttgg actgatcgag gccctgcgtg
     3361 ctgcgctggg tccgggaggg acgctcgtca tgccctcgtg gtcaggtctg gacgacgagc
     3421 cgttcgatcc tgccacgtcg cccgttacac cggaccttgg agttgtctct gacacattct
     3481 ggcgcctgcc aaatgtaaag cgcagcgccc atccatttgc ctttgcggca gcggggccac
     3541 aggcagagca gatcatctct gatccattgc ccctgccacc tcactcgcct gcaagcccgg
     3601 tcgcccgtgt ccatgaactc gatgggcagg tacttctcct cggcgtggga cacgatgcca
     3661 acacgacgct gcatcttgcc gagttgatgg caaaggttcc ctatggggtg ccgagacact
     3721 gcaccattct tcaggatggc aagttggtac gcgtcgatta tctcgagaat gaccactgct
     3781 gtgagcgctt tgccttggcg gacaggtggc tcaaggagaa gagccttcag aaggaaggtc
     3841 cagtcggtca tgcctttgct cggttgatcc gctcccgcga cattgtggcg acagccctgg
     3901 gtcaactggg ccgagatccg ttgatcttcc tgcatccgcc agaggcggga tgcgaagaat
     3961 gcgatgccgc tcgccagtcg attggctgag ctcataagtt cctattccga agttcctatt
     4021 ctctagaaag tataggaact tcgaagcagc tccagcctac acattaccct gttatcccta
     4081 ataatcgtcc acttgccgtc atgc
//
