LOCUS       sequence-210532-        3138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ORBIT integrating plasmid for C-terminal tagging with degradation 
            tag DAS+4, using zeocin as a selection marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3138)
  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 3138)
  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: pKM495 (Flag-Das+4) Zeo
FEATURES             Location/Qualifiers
     source          1..3138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(80..126)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    571..608
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             611..634
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      722..749
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      complement(916..1504)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(996..1015)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1769..1788)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        2537..2584
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2603..2977
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      3019..3105
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cgcagttcct ctgggggccg ccggacaccg cccccggcgc ctgacgcccc ctctagctga
       61 tcaccgcggc catgatggca taaaacgaaa ggcccagtct ttcgactgag cctttcgttt
      121 tatttgatgc ctggcagtcg atcgagaggt gaccacaacg acgcgcccgc tttgatcggg
      181 gacgtctgcg gccgaccatt tacgggtctt gttgtcgttg gcggtcatgg gccgaacata
      241 ctcacccgga tcggagggcc gaggacaagg tcgaacgagg ggcatgaccc ggtgcggggc
      301 ttcttgcact cggcataggc gagtgctaag aataacgttg gcactcgcga ccggtgagtg
      361 ctaggtcggg acggtgaggc caggcccgtc gtcgcagcga gtggcagcga ggacaacttg
      421 agccgtccgt cgcgggcact gcgcccggcc agcgtaagta gcggggttgc cgtcacccgg
      481 tgacccccgt ttcatccccg atccggagga atcacttcgc aatggccaag acaatgcgga
      541 tccatcgata gaaaggagga aggaaagctt ggcttgtcga cgacggcggt ctccgtcgtc
      601 aggatcatcg gactacaagg acgacgacga caaggccgcc aacgacgaga actactccga
      661 gaactacgcg gacgccagct agtgactcga gtctagagca tgcactagtg cggccgctag
      721 cagaaggcca tcctgacgga tggccttttc ttaagtcgcg ttgcgctcgg tcgttcggct
      781 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      841 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      901 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      961 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1021 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1081 tctcccctcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     1141 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     1201 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1261 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1321 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     1381 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1441 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1501 tcaagaagat cctttgatct tttctacggg gtctgacgct catggaacta gagcatgaga
     1561 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatcggcacg taagaggttc
     1621 caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt
     1681 ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat
     1741 atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta
     1801 taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca
     1861 caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt
     1921 ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac
     1981 cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt
     2041 ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta
     2101 tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt
     2161 caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat
     2221 gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca
     2281 tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga
     2341 tgagtggctg gcggaaacgg caatgctgaa gttttaaatc aatctaaagt atatatgagt
     2401 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2461 tatttcgttc atccatagtt gcctgactcc ccgacgtggc cgaccagccc gtcatcgtca
     2521 acgcctgacc gcggttgttg acaattaatc atcggcatag tatatcggca tagtataata
     2581 cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc
     2641 gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt
     2701 cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt
     2761 ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga
     2821 gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ctgggccggc
     2881 catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg
     2941 caactgcgtg cacttcgtgg ccgaggagca ggactgaatg catctctccc catgcgagag
     3001 tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     3061 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     3121 ttgaacgttg cgaactgg
//
