LOCUS       sequence-210459-        8698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For performing ORBIT - Expresses Che9c phage RecT and Bxb1 phage 
            Integrase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8698)
  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 8698)
  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: pKM444
FEATURES             Location/Qualifiers
     source          1..8698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      20..47
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             1533..3035
                     /codon_start=1
                     /transl_table=11
                     /gene="Bxb1"
                     /product="Bxb1 integrase"
                     /label=Bxb1 integrase
                     /note="Bxb1 large serine integrase from Mycobacterium 
                     bacteriophage Bxb1 (Ghosh et al. 2003)."
                     /protein_id="AFF61396.1"
                     /translation="MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLD
                     VSGAVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLV
                     VSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGY
                     LPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQG
                     REPQGREWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAE
                     LVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV
                     AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAG
                     SPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSM
                     NVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
     terminator      3070..3116
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3249..3268)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3260..4075
                     /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
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3333..3352)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(4136..4152)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4137..4153)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     CDS             7182..7805
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(7255..7274)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     rep_origin      7869..8457
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8358..8377
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8611..8628
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 actagttgat tagctaagca gaaggccatc ctgacggatg gcctttttgc gtttaatact
       61 gtttaaactc tagaaatatt ggatcgtcgg caccgtcacg gccgtgggag gcggcacgat
      121 ccgcgacgtg atgatcggcc gcatccccac ggtgctgcgc agtgagctct acgccatccc
      181 ggcgttgatc tgtgcgttcg cacgcacagg cccggtgtga gaagggtctc tgacgagcgg
      241 gagaactccc tatcagtgat agagtttgtc ctccctatca gtgatagata ggctctggga
      301 gtacccgtgt gtacgaccag cacggcatac atcatttcga cgccgagaga ttcgccgccc
      361 gaaatgagca cgatccgcat gcttaattaa gaaggagata tacatatggc tgaaaatgct
      421 gtcaccaagc aggattcgcc caaggcaccc gagacgatct cgcaagtgct gcaggtgctc
      481 gtgccgcagc tggcgcgtgc agtgcccaag ggtatggacc ccgaccgcat cgcgcggatc
      541 gtgcaaaccg agatccgcaa gtcgcgcaac gcgaaagctg ctggcatcgc taagcagtcc
      601 ctcgacgact gcacgcaaga gtcatttgcc ggtgcgctgc tgacctcggc cgcgctcggt
      661 ctcgagcccg gtgtcaacgg cgagtgctac ctcgtgccct accgcgacac ccggcgcggc
      721 gtggtcgagt gccagctgat catcggctac cagggcatcg tcaaactgtt ctggcagcac
      781 ccgcgcgcct cccggatcga cgcgcagtgg gtcggcgcga acgacgaatt ccattacacc
      841 atgggcctca atccgacgct gaaacacgtg aaggccaagg gtgatcgggg taatccggtc
      901 tacttctacg cgatcgtcga ggtgaccggc gctgagccgc tgtgggacgt gttcaccgcc
      961 gacgagatca gggaattgcg tcgcggcaag gtcggatcct cgggcgacat caaggacccg
     1021 cagcgctgga tggagcggaa gaccgcgctc aaacaggtgc tgaagctggc accgaagacg
     1081 acgcggctcg acgcggcgat ccgcgccgac gatcgcccgg gcaccgacct gtcacagtcg
     1141 caggcgctcg cgctgccgtc gaccgtcaag ccgacggccg actacatcga cggcgagatc
     1201 gccgagccgc acgaggtcga tacgccaccg aagtcgtcgc gcgcacaacg cgcgcagcgc
     1261 gccaccgcgc cggcgcccga cgtgcagatg gccaatcccg atcagctgaa gcgcctcggc
     1321 gagatccaga aggccgaaaa gtacaacgac gccgattggt tcaagttcct cgccgattcg
     1381 gccggcgtca aagccacgcg cgccgccgac ctcacgttcg acgaggcgaa ggctgtcatc
     1441 gacatgttcg acgggcccaa cgcatgagcg ccccggcgaa ttccgacgcg gtggttgatc
     1501 tgcgctagtg gaggcgccag aggcacttcg agatgagagc cctggtagtc atccgcctgt
     1561 cccgcgtcac cgatgctacg acttcaccgg agcgtcagct ggagtcttgc cagcagctct
     1621 gcgcccagcg cggctgggac gtcgtcgggg tagcggagga tctggacgtc tccggggcgg
     1681 tcgatccgtt cgaccggaag cgcagaccga acctggcccg gtggctagcg ttcgaggagc
     1741 aaccgttcga cgtgatcgtg gcgtaccggg tagaccggtt gacccgatcg atccggcatc
     1801 tgcagcagct ggtccactgg gccgaggacc acaagaagct ggtcgtctcc gcgaccgaag
     1861 cgcacttcga tacgacgacg ccgtttgcgg cggtcgtcat cgcgcttatg ggaacggtgg
     1921 cgcagatgga attagaagcg atcaaagagc ggaaccgttc ggctgcgcat ttcaatatcc
     1981 gcgccgggaa ataccgagga tccctgccgc cgtggggata cctgcctacg cgcgtggacg
     2041 gggagtggcg gctggtgccg gaccctgtgc agcgagagcg catcctcgag gtgtatcacc
     2101 gcgtcgtcga caaccacgag ccgctgcacc tggtggccca cgacctgaac cggcgtggtg
     2161 tcctgtcgcc gaaggactac ttcgcgcagc tgcaaggccg cgagccgcag ggccgggagt
     2221 ggtcggctac cgcgctgaag cgatcgatga tctccgaggc gatgctcggg tacgcgactc
     2281 tgaacggtaa gaccgtccga gacgacgacg gagccccgct ggtgcgggct gagccgatcc
     2341 tgacccgtga gcagctggag gcgctgcgcg ccgagctcgt gaagacctcc cgggcgaagc
     2401 ccgcggtgtc taccccgtcg ctgctgctgc gggtgttgtt ctgcgcggtg tgcggggagc
     2461 ccgcgtacaa gttcgccggg ggaggacgta agcacccgcg ctaccgctgc cgctcgatgg
     2521 ggttcccgaa gcactgcggg aacggcacgg tggcgatggc cgagtgggac gcgttctgcg
     2581 aggagcaggt gctggatctg ctcggggacg cggagcgtct ggagaaagtc tgggtagccg
     2641 gctcggactc cgcggtcgaa ctcgcggagg tgaacgcgga gctggtggac ctgacgtcgc
     2701 tgatcggctc cccggcctac cgggccggct ctccgcagcg agaagcactg gatgcccgta
     2761 ttgcggcgct ggccgcgcgg caagaggagc tggagggtct agaggctcgc ccgtctggct
     2821 gggagtggcg cgagaccggg cagcggttcg gggactggtg gcgggagcag gacaccgcgg
     2881 caaagaacac ctggcttcgg tcgatgaacg ttcggctgac gttcgacgtc cgcggcgggc
     2941 tgactcgcac gatcgacttc ggggatctgc aggagtacga gcagcatctc aggctcggca
     3001 gcgtggtcga acggctacac accgggatgt cgtaggttaa caactagcgt acgatcgact
     3061 gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatgcca
     3121 tcatggccgc gggacgtcgg ggggggggga aagccacgtt gtgtctcaaa atctctgatg
     3181 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     3241 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct cgaggccgcg
     3301 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     3361 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc catgcgccag agttgtttct
     3421 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     3481 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     3541 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     3601 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     3661 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     3721 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     3781 tgttgaacaa gtctggaaag aaatgcataa tcttttgcca ttctcaccgg attcagtcgt
     3841 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     3901 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     3961 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     4021 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     4081 tatcaagctt tagctaatta attggggacc ctagaggtcc ccttttttat tttatcgata
     4141 ccgtcgacct cgaggggggg cccccgggga tcatcgagcc gagaacgtta tcgaagttgg
     4201 tcatgtgtaa tcccctcgtt tgaactttgg attaagcgta gatacaccct tggacaagcc
     4261 agttggattc ggagacaagc aaattcagcc ttaaaaaggg cgaggccctg cggtggtgga
     4321 acaccgcagg gcctctaacc gctcgacgcg ctgcaccaac cagcccgcga acggctggca
     4381 gccagcgtaa ggcgcggctc atcgggcggc gttcgccacg atgtcctgca cttcgagcca
     4441 agcctcgaac acctgctggt gtgcacgact cacccggttg ttgacaccgc gcgcggccgt
     4501 gcgggctcgg tggggcggct ctgtcgccct tgccagcgtg agtagcgcgt acctcacctc
     4561 gcccaacagg tcgcacacag ccgattcgta cgccataaag ccaggtgagc ccaccagctc
     4621 cgtaagttcg ggcgctgtgt ggctcgtacc cgcgcattca ggcggcaggg ggtctaacgg
     4681 gtctaaggcg gcgtgtacgg ccgccacagc ggctctcagc ggcccggaaa cgtcctcgaa
     4741 acgacgcatg tgttcctcct ggttggtaca ggtggttggg ggtgctcggc tgtcgctggt
     4801 gttccaccac cagggctcga cgggagagcg ggggagtgtg cagttgtggg gtggcccctc
     4861 agcgaaatat ctgacttgga gctcgtgtcg gaccatacac cggtgattaa tcgtggtcta
     4921 ctaccaagcg tgagccacgt cgccgacgaa tttgagcagc tctggctgcc gtactggccg
     4981 ctggcaagcg acgatctgct cgaggggatc taccgccaaa gccgcgcgtc ggccctaggc
     5041 cgccggtaca tcgaggcgaa cccaacagcg ctggcaaacc tgctggtcgt ggacgtagac
     5101 catccagacg cagcgctccg agcgctcagc gcccgggggt cccatccgct gcccaacgcg
     5161 atcgtgggca atcgcgccaa cggccacgca cacgcagtgt gggcactcaa cgcccctgtt
     5221 ccacgcaccg aatacgcgcg gcgtaagccg ctcgcataca tggcggcgtg cgccgaaggc
     5281 cttcggcgcg ccgtcgacgg cgaccgcagt tactcaggcc tcatgaccaa aaaccccggc
     5341 cacatcgcct gggaaacgga atggctccac tcagatctct acacactcag ccacatcgag
     5401 gccgagctcg gcgcgaacat gccaccgccg cgctggcgtc agcagaccac gtacaaagcg
     5461 gctccgacgc cgctagggcg gaattgcgca ctgttcgatt ccgtcaggtt gtgggcctat
     5521 cgtcccgccc tcatgcggat ctacctgccg acccggaacg tggacggact cggccgcgcg
     5581 atctatgccg agtgccacgc gcgaaacgcc gaattcccgt gcaacgacgt gtgtcccgga
     5641 ccgctaccgg acagcgaggt ccgcgccatc gccaacagca tttggcgttg gatcacaacc
     5701 aagtcgcgca tttgggcgga cgggatcgtg gtctacgagg ccacactcag tgcgcgccag
     5761 tcggccatct cgcggaaggg cgcagcagcg cgcacggcgg cgagcacagt tgcgcggcgc
     5821 gcaaagtccg cgtcagccat ggaggcattg ctatgagcga cggctacagc gacggctaca
     5881 accggcagcc gactgtccgc aaaaagcggc gcgtgaccgc cgccgaaggc gctcgaatca
     5941 ccggactatc cgaacgccac gtcgtccggc tcgtggcgca ggaacgcagc gagtggctcg
     6001 ccgagcaggc tgcacgccgc gaacgcatcc gcgcctatca cgacgacgag ggccactctt
     6061 ggccgcaaac ggccaaacat ttcgggctgc atctggacac cgttaagcga ctcggctatc
     6121 gggcgaggaa agagcgtgcg gcagaacagg aagcggctca aaaggcccac aacgaagccg
     6181 acaatccacc gctgttctaa cgcaattggg gagcgggtgt cgcgggggtt ccgtgggggg
     6241 ttccgttgca acgggtcgga caggtaaaag tcctggtaga cgctagtttt ctggtttggg
     6301 ccatgcctgt ctcgttgcgt gtttcgttgc gcccgttttg aataccagcc agacgagacg
     6361 gggttctacg aatcttggtc gataccaagc catttccgct gaatatcggg gagctcaccg
     6421 ccagaatcgg tggttgtggt gatgtacgtg gcgaactccg ttgtagtgcc tgtggtggca
     6481 tccgtggcca ctctcgttgc acggttcgtt gtgccgttac aggccccgtt gacagctcac
     6541 cgaacgtagt taaaacatgc tggtcaaact aggtttacca acgatacgag tcagctcatc
     6601 tagggccagt tctaggcgtt gttcgttgcg cggttcgttg cgcatgtttc gtgtggttgc
     6661 tagatggctc cgcaaccaca cgcttcgagg ttgagtgctt ccagcacggg cgcgatccag
     6721 aagaacttcg tcgtgcgact gtcctcgttg atccttgccg agctgggatg gaagcccggc
     6781 cgacccaccc tggaggagat gatcgaggat gccagggcct ttcacgcccg ccgctgctga
     6841 gcgtccgccg ccgggcccgc accgccgtcg gccggcccgc tccgggctcg cagcagcggg
     6901 cttcggcgcg ggcccggggc tcccgggccg ccgggcgggg ctccgcccgg cggccgctga
     6961 ttagctaagc agaaggccat cctgacggat gcctttttcg ctttaatact gcatgcactc
     7021 tagaaatatt ggatcgtcgc accgggttaa gcctggagtg cggtggtgcc tggtcggcat
     7081 tttcgcagtc gagggctctc gtgtagcctg ggcgagttgc cgacgcaggc gaccctcctg
     7141 ccacggatcc gcatggtacc atcaggagga atcacttcgc aatgtctaga ttagataaaa
     7201 gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa
     7261 cccgtaaact cgcccagaag cttggtgtag agcagcctac attgtattgg catgtaaaaa
     7321 ataagcgggc cctgctcgac gccttagcca ttgagatgtt agataggcac catactcact
     7381 tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta
     7441 gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag
     7501 aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac
     7561 tagagaacgc gttatatgca ctcagcgctg tggggcattt taccttaggt tgcgtattgg
     7621 aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc
     7681 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct
     7741 tattcggcct tgaattgatc atctgcggat tagaaaaaca acttaaatgt gaaagtgggt
     7801 cttaatgacc atgggctaga tttaaatcac tgagcgtcag accccgtaga aaagatcaaa
     7861 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     7921 ccgctaccag cggtggtttt tttgccggat caagagctac caactctttt tccgaaggta
     7981 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     8041 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     8101 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     8161 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     8221 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     8281 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     8341 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     8401 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     8461 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     8521 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     8581 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     8641 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctaga
//
