LOCUS       sequence-418043-        4246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ORBIT integrating plasmid for C-terminal tagging with cleavable 
            EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4246)
  AUTHORS   Murphy KC, Nelson SJ, Nambi S, Papavinasasundaram K, Baer CE, 
            Sassetti CM
  TITLE     ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial 
            Chromosomes.
  JOURNAL   MBio. 2018 Dec 11;9(6). pii: mBio.01467-18. doi: 
            10.1128/mBio.01467-18.
  PUBMED    30538179
REFERENCE   2  (bases 1 to 4246)
  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: "MBio."; 
            date: "2018-12-11"; volume: "9(6). pii: mBio.01467-18. doi"; pages: 
            " 10.1128/mBio.01467-18"
COMMENT     Sequence Label: pKM493
FEATURES             Location/Qualifiers
     source          1..4246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    468..505
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             532..555
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(568..570,571..573,574..1284)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(613..634)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(874..893)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1221..1242
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      1303..1330
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1497..2085)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1577..1596)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2350..2369)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(join(4222..4246,1..22))
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 actgagcctt tcgttttatt tgatgcctgg cagtcgatcg agaggtgacc acaacgacgc
       61 gcccgctttg atcggggacg tctgcggccg accatttacg ggtcttgttg tcgttggcgg
      121 tcatgggccg aacatactca cccggatcgg agggccgagg acaaggtcga acgaggggca
      181 tgacccggtg cggggcttct tgcactcggc ataggcgagt gctaagaata acgttggcac
      241 tcgcgaccgg tgagtgctag gtcgggacgg tgaggccagg cccgtcgtcg cagcgagtgg
      301 cagcgaggac aacttgagcc gtccgtcgcg ggcactgcgc ccggccagcg taagtagcgg
      361 ggttgccgtc acccggtgac ccccgtttca tccccgatcc ggaggaatca cttcgcaatg
      421 gccaagacaa ttgcggatcc atcgatagaa aggaggaagg aaagcttggc ttgtcgacga
      481 cggcggtctc cgtcgtcagg atcatctcga gcggagaacc tgtacttcca ggactacaag
      541 gacgacgacg acaagggcgg cggcggcatg gtgtcgaagg gcgaggagct gttcaccggc
      601 gtcgtgccca tcctggtcga gctggacggc gacgtgaacg gccacaagtt cagcgtctcg
      661 ggcgagggcg agggcgacgc gacctacggc aagctgaccc tgaagttcat ctgcaccacc
      721 ggcaagctgc cggtcccctg gccgaccctg gtgaccaccc tgacctacgg cgtgcagtgt
      781 ttctcgcggt accccgacca catgaagcag cacgacttct tcaagagcgc gatgccggag
      841 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgggcc
      901 gaggtcaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
      961 aaggaagacg gcaacatcct gggccacaag ctggagtaca actacaactc gcacaacgtc
     1021 tacatcatgg cggacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1081 atcgaggacg gcagcgtcca gctggccgac cactaccagc agaacacccc catcggcgac
     1141 ggccccgtcc tgctgccgga caaccactac ctgagcaccc agtcggcgct gagcaaggac
     1201 ccgaacgaga agcgggacca catggtcctg ctggagttcg tgaccgccgc gggcatcacc
     1261 ctgggcatgg acgagctgta caagtagtga gcggccgcta gcagaaggcc atcctgacgg
     1321 atggcctttt cttaagtcgc gttgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1381 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1441 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1501 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1561 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1621 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccctc gggaagcgtg
     1681 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1741 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1801 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1861 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1921 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1981 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2041 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2101 ttttctacgg ggtctgacgc tcatggaact agagcatgag attatcaaaa aggatcttca
     2161 cctagatcct tttaaattaa aaatcggcac gtaagaggtt ccaactttca ccataatgaa
     2221 ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct
     2281 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     2341 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     2401 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     2461 attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac
     2521 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact
     2581 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata
     2641 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt
     2701 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac
     2761 gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa
     2821 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     2881 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggct ggcggaaacg
     2941 gcaatgctga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3001 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3061 tgcctgactc cccgacgtgg ccgaccagcc cgtcatcgtc aacgcctgat ccgcggtgcg
     3121 gacaggccgt gtcgcgaccg gccgtgcgga attaagccgg cccgtaccct gtgaatagag
     3181 gtccgctgtg acacaagaat ccctgttact tctcgaccgt attgattcgg atgattccta
     3241 cgcgagcctg cggaacgacc aggaattctg ggagccgctg gcccgccgag ccctggagga
     3301 gctcgggctg ccggtgccgc cggtgctgcg ggtgcccggc gagagcacca accccgtact
     3361 ggtcggcgag cccggcccgg tgatcaagct gttcggcgag cactggtgcg gtccggagag
     3421 cctcgcgtcg gagtcggagg cgtacgcggt cctggcggac gccccggtgc cggtgccccg
     3481 cctcctcggc cgcggcgagc tgcggcccgg caccggagcc tggccgtggc cctacctggt
     3541 gatgagccgg atgaccggca ccacctggcg gtccgcgatg gacggcacga ccgaccggaa
     3601 cgcgctgctc gccctggccc gcgaactcgg ccgggtgctc ggccggctgc acagggtgcc
     3661 gctgaccggg aacaccgtgc tcacccccca ttccgaggtc ttcccggaac tgctgcggga
     3721 acgccgcgcg gcgaccgtcg aggaccaccg cgggtggggc tacctctcgc cccggctgct
     3781 ggaccgcctg gaggactggc tgccggacgt ggacacgctg ctggccggcc gcgaaccccg
     3841 gttcgtccac ggcgacctgc acgggaccaa catcttcgtg gacctggccg cgaccgaggt
     3901 caccgggatc gtcgacttca ccgacgtcta tgcgggagac tcccgctaca gcctggtgca
     3961 actgcatctc aacgccttcc ggggcgaccg cgagatcctg gccgcgctgc tcgacggggc
     4021 gcagtggaag cggaccgagg acttcgcccg cgaactgctc gccttcacct tcctgcacga
     4081 cttcgaggtg ttcgaggaga ccccgctgga tctctccggc ttcaccgatc cggaggaact
     4141 ggcgcagttc ctctgggggc cgccggacac cgcccccggc gcctgacgcc ccctctagct
     4201 gatcaccgcg gccatgatgg cataaaacga aaggcccagt ctttcg
//
