LOCUS       sequence-210461-        3878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ORBIT integrating plasmid for C-terminal tagging with EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3878)
  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 3878)
  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: pKM468-EGFP
FEATURES             Location/Qualifiers
     source          1..3878
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    47..84
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             join(87..89,90..92,93..803)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(132..153)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(393..412)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     740..761
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             834..857
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             858..875
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     rep_origin      complement(1101..1689)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1181..1200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1954..1973)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(3826..3872)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ggcagtcgat cgtacgctag ttaactacgt cgacatcgat aagcttggct tgtcgacgac
       61 ggcggtctcc gtcgtcagga tcatcgatgg tgtcgaaggg cgaggagctg ttcaccggcg
      121 tcgtgcccat cctggtcgag ctggacggcg acgtgaacgg ccacaagttc agcgtctcgg
      181 gcgagggcga gggcgacgcg acctacggca agctgaccct gaagttcatc tgcaccaccg
      241 gcaagctgcc ggtcccctgg ccgaccctgg tgaccaccct gacctacggc gtgcagtgtt
      301 tctcgcggta ccccgaccac atgaagcagc acgacttctt caagagcgcg atgccggagg
      361 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgggccg
      421 aggtcaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
      481 aggaagacgg caacatcctg ggccacaagc tggagtacaa ctacaactcg cacaacgtct
      541 acatcatggc ggacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
      601 tcgaggacgg cagcgtccag ctggccgacc actaccagca gaacaccccc atcggcgacg
      661 gccccgtcct gctgccggac aaccactacc tgagcaccca gtcggcgctg agcaaggacc
      721 cgaacgagaa gcgggaccac atggtcctgc tggagttcgt gaccgccgcg ggcatcaccc
      781 tgggcatgga cgagctgtac aagggcggcg gcggcgagaa cctgtacttc caggactaca
      841 aggacgacga cgacaagcac caccaccacc accactagtg actcgagtct agagcatgca
      901 ctagtgcggc cgctagcggt accagatctg ggcccttaag tcgcgttgcg ctcggtcgtt
      961 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     1021 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1081 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1141 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1201 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1261 gcctttctcc cctcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1321 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1381 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1441 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1501 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     1561 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1621 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1681 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcatgg aactagagca
     1741 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatcg gcacgtaaga
     1801 ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg
     1861 agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt
     1921 gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt
     1981 acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat
     2041 aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg
     2101 gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt
     2161 tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac
     2221 gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg
     2281 gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg
     2341 agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc
     2401 accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt
     2461 catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac
     2521 tgcgatgagt ggctggcgga aacggcaatg ctgaagtttt aaatcaatct aaagtatata
     2581 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2641 ctgtctattt cgttcatcca tagttgcctg actccccgac gtggccgacc agcccgtcat
     2701 cgtcaacgcc tgatccgcgg tgcggacagg ccgtgtcgcg accggccgtg cggaattaag
     2761 ccggcccgta ccctgtgaat agaggtccgc tgtgacacaa gaatccctgt tacttctcga
     2821 ccgtattgat tcggatgatt cctacgcgag cctgcggaac gaccaggaat tctgggagcc
     2881 gctggcccgc cgagccctgg aggagctcgg gctgccggtg ccgccggtgc tgcgggtgcc
     2941 cggcgagagc accaaccccg tactggtcgg cgagcccggc ccggtgatca agctgttcgg
     3001 cgagcactgg tgcggtccgg agagcctcgc gtcggagtcg gaggcgtacg cggtcctggc
     3061 ggacgccccg gtgccggtgc cccgcctcct cggccgcggc gagctgcggc ccggcaccgg
     3121 agcctggccg tggccctacc tggtgatgag ccggatgacc ggcaccacct ggcggtccgc
     3181 gatggacggc acgaccgacc ggaacgcgct gctcgccctg gcccgcgaac tcggccgggt
     3241 gctcggccgg ctgcacaggg tgccgctgac cgggaacacc gtgctcaccc cccattccga
     3301 ggtcttcccg gaactgctgc gggaacgccg cgcggcgacc gtcgaggacc accgcgggtg
     3361 gggctacctc tcgccccggc tgctggaccg cctggaggac tggctgccgg acgtggacac
     3421 gctgctggcc ggccgcgaac cccggttcgt ccacggcgac ctgcacggga ccaacatctt
     3481 cgtggacctg gccgcgaccg aggtcaccgg gatcgtcgac ttcaccgacg tctatgcggg
     3541 agactcccgc tacagcctgg tgcaactgca tctcaacgcc ttccggggcg accgcgagat
     3601 cctggccgcg ctgctcgacg gggcgcagtg gaagcggacc gaggacttcg cccgcgaact
     3661 gctcgccttc accttcctgc acgacttcga ggtgttcgag gagaccccgc tggatctctc
     3721 cggcttcacc gatccggagg aactggcgca gttcctctgg gggccgccgg acaccgcccc
     3781 cggcgcctga cgccccctct agctgatcac cgcggccatg atggcataaa acgaaaggcc
     3841 cagtctttcg actgagcctt tcgttttatt tgatgcct
//
