LOCUS       sequence-262707-        6842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An integrative plasmid able to introduce a second copy of the pip 
            gene in the chromosome of our conditional mutants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6842)
  AUTHORS   Boldrin F, Anoosheh S, Serafini A, Cioetto Mazzabo L, Palu G, 
            Provvedi R, Manganelli R
  TITLE     Improving the stability of the TetR/Pip-OFF mycobacterial 
            repressible promoter system.
  JOURNAL   Sci Rep. 2019 Apr 8;9(1):5783. doi: 10.1038/s41598-019-42319-2.
  PUBMED    30962489
REFERENCE   2  (bases 1 to 6842)
  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: "Sci Rep."; 
            date: "2019-04-8"; volume: "9(1):5783. doi"; pages: " 
            10.1038/s41598-019-42319-2"
COMMENT     Sequence Label: pAGN33
FEATURES             Location/Qualifiers
     source          1..6842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(187..642)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(324..345)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     536..555
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     840..862
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     854..871
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     855..871
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1613..1624
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3710..3729)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3721..4536
                     /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
                     SDFDDERNGWPVEQVWKEMHKFLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3794..3813)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(4622..4638)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4622..4638)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4635..4657)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4646..4662
                     /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)."
     promoter        complement(join(4670..4676,4677..4694,4695..4700))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4715..4736
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4853..4870)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5024..5611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5104..5123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5782..6573,6574..6642))
                     /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     6405..6424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6643..6747)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6815..6833
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
      241 gctcattttt taaccaatag accgaaatcg gcaaaatccc ttataaatca aaagaatagc
      301 ccgagataga gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      361 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      421 cacccaaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      481 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      541 agaaagcgaa aggagcgggc gctaaggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      601 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg
      661 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
      721 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
      781 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
      841 ccagtcacga cgttgtaaaa cgacggccag tgaattagct tgatatccca tcctgacgga
      901 tggccttcca ccatgcaggc ccggctcatc ggaacatacg aaggctgatc cacccgccat
      961 cccacgatcc agcggccccg gggcgatcgg gtcctagcag acgcctccct atcagtgata
     1021 gatcaactga tccctatcag tgatagacat attgtctagt gtggcggccg cgagtcgagg
     1081 agaggtgcgc atggcgaagg cagggcggga ggggccgcgg gacagcgtgt ggctgtcggg
     1141 ggaggggcgg cgcggcggtc gccgtggggg gcagccgtcc gggctcgacc gggaccggat
     1201 caccggggtc accgtccggc tgctggacac ggagggcctg acggggttct cgatgcgccg
     1261 cctggccgcc gagctgaacg tcaccgcgat gtccgtgtac tggtacgtcg acaccaagga
     1321 ccagttgctc gagctcgccc tggacgccgt cttcggcgag ctgcgccacc cggacccgga
     1381 cgccgggctc gactggcgcg aggaactgcg ggccctggcc cgggagaacc gggcgctgct
     1441 ggtgcgccac ccctggtcgt cccggctggt cggcacctac ctcaacatcg gcccgcactc
     1501 gctggccttc tcccgcgcgg tgcagaacgt cgtgcgccgc agcgggctgc ccgcgcaccg
     1561 cctgaccggc gccatctcgg ccgtcttcca gttcgtctac ggctacggca ccatcgaggg
     1621 ccgcttcctc gcccgggtgg cggacaccgg gctgagtccg gaggagtact tccaggactc
     1681 gatgaccgcg gtgaccgagg tgccggacac cgcgggcgtc atcgaggacg cgcaggacat
     1741 catggcggcc cggggcggcg acaccgtggc ggagatgctg gaccgggact tcgagttcgc
     1801 cctcgacctg ctcgtcgcgg gcatcgacgc gatggtcgaa caggcctgat taattaaaag
     1861 ctaattcgag ctcggtaccc ggggatcctc tagagtcgag atttacagtc ggtcgcctac
     1921 gcaaacgtcg atgggagcag ttcggacagc ccctgcatgg cctccagatg cctcgcccga
     1981 tccgcatgcg catagatccg ctgcgcatcc acactcgcat gacccaagat ctccatacgc
     2041 gtttgctcat ccacacccgc tgcgcgcagc aatgtcgagg tggtgtgccg cgagttgtgc
     2101 ggcggtaacg actcggttgg accgatcacc ccagcagcgc ggaacacgcc acgccacacg
     2161 tcgtagtccg aacggggatc gatcggcttc ccctccttgt gccacaccaa gtcatgcgga
     2221 ttgtcggtgc ggagtttctg catcgccaca tacaacggcg gcaacaacgg aacctcacgc
     2281 caaccagcgt ccgtcttcgg ccgggtgaac aacaacgacc cctcacattc ctgatactcg
     2341 aaatgcgccg gcaggtccca ccgggactgc gggcatgccc atgcccgtgt cttcccgcaa
     2401 ggccagtacg ggggtttctt gggcatacgg tcgggccggg acagcggtga cggttcgggt
     2461 agaggatccc cacagccgtg gacgcgggtt tccgattgca actgccaagc gatggtgatc
     2521 catccctgag cggggttgtc gacgtagggc cagcgcaggc cgaggagttc cccacggcgg
     2581 gcgcccgtca ggaaaccggc ggcgatccgc accgcatccg gttcgtcgca cacctggaac
     2641 gcggtgtgaa tgatgtgctg cgccacgtcc gccgggaagc cgttgcgttt cttcttccgg
     2701 tactcgggct tgtcgaccaa tgcggccaca ttcctggtcg ccacaccctc cgctaccgca
     2761 tcgtccaagg ctttctggac gatgacatgg accagctcgg cggtgcggga ggccccgatc
     2821 tcggagtgca ggtcccgcac atgctgcggg gtgagtttgt cgatgcgttt cgcgccgagg
     2881 atcgggttga tgtggttgtg gatggcggcc cggtagtcgt tgaggacgcc ggggcggact
     2941 ttacgtttgg cgtggatgtt gtcgatccag tgcagcatcc acttctccac agttgtggat
     3001 gaggtggtgg cgatgcggcc ctcttcgacg tcgcggcgga gttgtttgag tttggccatg
     3061 gcggtgttgc ggtcaacgga ggacacccat ttgtagcggc ggttgccgtt gcggtctggg
     3121 ggtagttcta ctcgtcccat ccatttgccg tcggcgcgtt ggaagaacgc tccgtctccg
     3181 cgggttctgc gtttcttagt tgccatcgtt ttccctccca gggggtcacc ctacggttca
     3241 ccctacggtg ctgcgcagca ttacgcagaa ttgcgcagta tcgggggtct acctgcgggt
     3301 ttgacaacgt ttctcctggt atgcagccta tcaaccgctg actcttaatc agcgggtcgg
     3361 gggttcgaaa ccctcacggc gcacaggtca gaggccataa gcctcagagg ggatcaccct
     3421 aaaggtaacc ctataggggt tttcactggt cctacatgtc gtcgcgccgt cgtactctct
     3481 tttcataggg gagcatgagg atccaactga gcttgctgtg ccttcgattg agcggttgaa
     3541 gcatctggag cgtcggcgga cagcgatcac ggttcccaac gatggctgaa agaaggatga
     3601 aatgactgca ctcgacctgc aggcatgcaa gcttggccgg gttacattgc acaagataaa
     3661 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     3721 atgagccata ttcaacggga aacgtcttgc tcgaggccgc gattaaattc caacatggat
     3781 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc
     3841 tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc
     3901 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct
     3961 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg
     4021 atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt
     4081 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct
     4141 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg
     4201 gttgatgcga gtgattttga tgacgagcgt aatggttggc ctgttgaaca agtctggaaa
     4261 gaaatgcata agtttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca
     4321 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc
     4381 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     4441 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     4501 ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa
     4561 cactggcaga gcattacgct gacttgacgg gacggcgccg gccaagcttg gcgtaatcat
     4621 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4681 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4741 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4801 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4861 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4921 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4981 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5041 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5101 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5161 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5221 gctcacgctg tagtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5281 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5341 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5401 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5461 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5521 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5581 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5641 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5701 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5761 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5821 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5881 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5941 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6001 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6061 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6121 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6181 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6241 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6301 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6361 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6421 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6481 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6541 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6601 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6661 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6721 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     6781 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     6841 tc
//
