LOCUS       sequence-229899-        5312 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generation of unmarked gene deletions in Mycobacterium smegmatis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5312)
  AUTHORS   Shenkerman Y, Elharar Y, Vishkautzan M, Gur E
  TITLE     Efficient and simple generation of unmarked gene deletions in 
            Mycobacterium smegmatis.
  JOURNAL   Gene. 2014 Jan 1;533(1):374-8. doi: 10.1016/j.gene.2013.09.082. Epub
            2013 Oct 4.
  PUBMED    24100088
REFERENCE   2  (bases 1 to 5312)
  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"; doi: 
            "10.1016/j.gene.2013.09.082"; journalName: "Gene"; date: "2014-01-1-
            1"; volume: "533"; issue: "1"; pages: "374-8"
COMMENT     Sequence Label: pYS2
FEATURES             Location/Qualifiers
     source          1..5312
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    615..648
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3056..3089
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3141..3160)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3260..3282
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3320..3338)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3406..3510
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3511..3579,3580..4371)
                     /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(3729..3748)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4542..5130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5031..5050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5284..5301
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga attcgagctc
      241 ggtacccggg gatcctcggt cgacgtgtgc gcgtagccgc tgcgccagaa cacccgcatc
      301 gcggtgtcca gtgcgacgtc cggatcgaac tccttgtgac gggccatgtg ccaattctag
      361 atcgtcgaga aggtcgacgg ccagtggagg acgtactgct ccgagacctg ctactggacc
      421 gacgccgtcg cgttccgggg tgagtacgag ggcagggaaa ccccgaacat gggacggctc
      481 accgggttcc gagaatggga gaccctgcac cacggcaatc caatattgat ccactagttt
      541 aaaccaagaa gatcctttga tattttctac ggggtctgac gctcagtcga gtatacgagg
      601 cgcgccagat ttaaataact tcgtataatg tatgctatac gaagttatcc cagatctgta
      661 agataaactc tagaaatatt ggatcgtcgg caccgtcacg gccgtgggag gcggcacgat
      721 ccgcgacgtg atgatcggcc gcatccccac ggtgctgcgc agtgagctct acgccatccc
      781 ggcgttgatc tgtgcgttcg cacgcacagg cccggtgtga gaagggtctc tgacgagcgg
      841 gagaacccac ccggggtggg cgagtttgtc ctgcgtgtgc tcggtcgagt aggctctggg
      901 agtacccgtg tgtacgacca gcacggcata catcatttcg acgccgagag attcgccgcc
      961 cgaaatgagc acgatccgca tgcttaacag aaaggaggtt aataatgtcg aagggcgagg
     1021 agctgttcac cggcgtcgtc ccgatcctgg tcgagctgga cggtgacgtc aacggccaca
     1081 agttctccgt ctccggcgag ggtgagggcg acgccaccta cggcaagctg accctgaagt
     1141 tcatctgcac caccggtaag ctgccggtcc cgtggccgac cctggtcacc accctgacct
     1201 acggcgtcca gtgcttctcc cgctacccgg accacatgaa gcgccacgac ttcttcaagt
     1261 ccgccatgcc ggagggttac gtccaggagc gcaccatctc cttcaaggac gacggtaact
     1321 acaagacgcg tgccgaggtc aagttcgagg gcgacaccct ggtcaaccgc atcgagctga
     1381 agggcatcga cttcaaggag gacggtaaca tcctgggcca caagctggag tacaactaca
     1441 actcccacaa cgtctacatc accgcggaca agcagaagaa cggcatcaag gccaacttca
     1501 agacccgcca caacatcgag gacggtggcg tccagctagc cgaccactac cagcagaaca
     1561 ccccgatcgg cgacggcccg gtcctgctgc cggacaacca ctacctgtcc acccagtccg
     1621 ccctgtccaa ggacccgaac gagaagcgcg accacatggt cctgctggag ttcgtcaccg
     1681 ccgccggcat cacccacggc atggacgagc tgtacaagta gatttatcac cagcccgtca
     1741 tcgtcaacgc ctgatccgcg gtgcggacag gccgtgtcgc gaccggccgt gcggaattaa
     1801 gccggcccgt accctgtgaa tagaggtccg ctgtgacaca agaatccctg ttacttctcg
     1861 accgtattga ttcggatgat tcctacgcga gcctgcggaa cgaccaggaa ttctgggagc
     1921 cgctggcccg ccgagccctg gaggagctcg ggctgccggt gccgccggtg ctgcgggtgc
     1981 ccggcgagag caccaacccc gtactggtcg gcgagcccgg cccggtgatc aagctgttcg
     2041 gcgagcactg gtgcggtccg gagagcctcg cgtcggagtc ggaggcgtac gcggtcctgg
     2101 cggacgcccc ggtgccggtg ccccgcctcc tcggccgcgg cgagctgcgg cccggcaccg
     2161 gagcctggcc gtggccctac ctggtgatga gccggatgac cggcaccacc tggcggtccg
     2221 cgatggacgg cacgaccgac cggaacgcgc tgctcgccct ggcccgcgaa ctcggccggg
     2281 tgctcggccg gctgcacagg gtgccgctga ccgggaacac cgtgctcacc ccccattccg
     2341 aggtcttccc ggaactgctg cgggaacgcc gcgcggcgac cgtcgaggac caccgcgggt
     2401 ggggctacct ctcgccccgg ctgctggacc gcctggagga ctggctgccg gacgtggaca
     2461 cgctgctggc cggccgcgaa ccccggttcg tccacggcga cctgcacggg accaacatct
     2521 tcgtggacct ggccgcgacc gaggtcaccg ggatcgtcga cttcaccgac gtctatgcgg
     2581 gagactcccg ctacagcctg gtgcaactgc atctcaacgc cttccggggc gaccgcgaga
     2641 tcctggccgc gctgctcgac ggggcgcagt ggaagcggac cgaggacttc gcccgcgaac
     2701 tgctcgcctt caccttcctg cacgacttcg aggtgttcga ggagaccccg ctggatctct
     2761 ccggcttcac cgatccggag gaactggcgc agttcctctg ggggccgccg gacaccgccc
     2821 ccggcgcctg acgccccggg ccgcccggcg ccgcccccgg cccccggcgg ccatcgaccc
     2881 aagtaccgcc acctaacaat tcgttcaagc cgagatcggc ttcccggccg cggagttgtt
     2941 cggtaaattg tcacaacgaa ttcgataaaa taaaaaaggg gacctctagg gtcccctttt
     3001 ttattttatc gaattcgagc tcgaagttcc tattctctag aaagcttgaa cgcgtataac
     3061 ttcgtataat gtatgctata cgaagttatt aattaaagct agcacatgca tggttattgt
     3121 ctcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3181 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3241 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3301 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3361 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3421 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3481 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3541 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3601 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3661 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3721 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3781 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3841 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3901 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3961 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4021 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4081 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4141 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4201 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4261 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4321 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4381 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4441 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4501 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4561 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4621 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4681 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4741 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4801 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4861 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4921 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4981 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5041 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5101 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5161 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5221 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     5281 cgcagcgagt cagtgagcga ggaagcggaa ga
//
