LOCUS       62056_17480        5973 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT445, complete sequence.
ACCESSION   MN991277
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5973)
  AUTHORS   Taketani M, Zhang J, Zhang S, Triassi AJ, Huang YJ, Griffith LG, 
            Voigt CA.
  TITLE     Genetic circuit design automation for the gut resident species 
            Bacteroides thetaiotaomicron
  JOURNAL   Nat. Biotechnol. (2020) In press
  PUBMED    32231334
REFERENCE   2  (bases 1 to 5973)
  AUTHORS   Taketani M, Voigt CA.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-JAN-2020) Biological Engineering, MIT, Synthetic 
            Biology Center 500 Technology Square NE47-140, Cambridge, MA 02139, 
            USA
REFERENCE   3  (bases 1 to 5973)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Biotechnol. (2020) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (25-JAN-2020) Biological Engineering, MIT, Synthetic Biology Center 
            500 Technology Square NE47-140, Cambridge, MA 02139, USA"
FEATURES             Location/Qualifiers
     source          1..5973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41648.1"
                     /translation="MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFT
                     AGLVKRCNFVTAIEIDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPY
                     NISTNIIRKIVFESSATISYLIVEYGFAKMLLDTNRSLALLLMAEVDISILAKIPRYYF
                     HPKPKVDSTLIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQFNKALKHARIY
                     DINNISFEQFVSLFNSYKIFNG"
     CDS             1378..2235
                     /label=AmpR
                     /note="beta-lactamase"
     oriT            complement(2369..2478)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      2649..3037
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     regulatory      3078..3107
                     /label=tL17
                     /note="tL17"
                     /regulatory_class="terminator"
     regulatory      3123..3130
                     /label=L3S3P21
                     /note="L3S3P21"
                     /regulatory_class="terminator"
     protein_bind    complement(3222..3240)
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3284..3302
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        3308..3358
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3382..3424
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3425..3937
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      3957..4017
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     primer_bind     complement(4143..4163)
                     /label=pNBU1_seq.RE
                     /note="pNBU1_seq.RE"
     misc_recomb     4261..4590
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    4388..4401
                     /label=CC Region
                     /note="CC Region"
     misc_difference 4400
                     /replace="g"
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             4606..5943
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41651.1"
                     /translation="MKVTFIIKKAAKRYDTESMATIYVRFRNGRQLDSVAPTQLAINPN
                     LWDDKDECVKTKAVCNEEMRTHINEEIRQLKTYIEKVYQQEKEAIDKEWLKTTLDKFYH
                     PEKYFLPEEVVIKPTIGELFDEFLNKHPLSEVRKKNFRVVKRALLRYELYVRATKRGQK
                     GFILDVDLVTPDTLRDMWDFFQNEYQYYELYPSIYEAIPEKRTPQPRSKNTLIDCFSRI
                     RTFFLWCFDNKRTTNRPFDKFPIEECTYGTPYYITLEERDRIFNADLSATPQLAIQRDI
                     FIFQTLIGCRVSDLYRMTKLNVVNEAIEYIPKKTKEGNPVTVRVPLNDKAKEILERYKE
                     YEGKLLPFISEQKYNDAIKKIFKLAGVDRIVTILDPLTHNEIKRPIYEVASSHLARRTF
                     IGNIYKKVKDPNLVSALSGHKEGSKAFRRYRDIDEEMKKDLVKLLD"
ORIGIN
        1 agagtagcga tattaaaaga atccgatgag aaaagactaa tatttatcta tccattcagt
       61 ttgatttttc aggactttac atcgtcctga aagtatttgt tggtaccggt accgaggacg
      121 cgtaaacatt tacagttgca tgtggcctat tgtttttagc cgttaaatat tttataacta
      181 ttaaatagcg atacaaattg ttcgaaacta atattgttta tatcatatat tctcgcatgt
      241 tttaaagctt tattaaattg attttttgta aacagttttt cgtactcttt gttaacccat
      301 ttcattacaa aagtttcata tttttttctc tctttaaatg ccatttttgc tggctttctt
      361 tttaatacaa ttaatgtgct atccacttta ggttttggat ggaaataata cctaggaatt
      421 tttgctaata tagaaatatc tacctctgcc attaacagca atgctagtga tctgtttgta
      481 tctaataaca ttttagcaaa accatattcc actattaaat aacttattgt ggctgaactt
      541 tcaaaaacaa tttttcgaat tatatttgtg cttatgttgt aaggtatgct gccaaatatt
      601 ttatatggat tgtggctagg aaatgtaaat ttcagtatat catcatttac tatttgatag
      661 ttaggataat ttaagagctt attacgagtt acctcacata atttagaatc aatttctatc
      721 gccgttacaa aattacatct ctttaccaat ccagcagtaa aatgaccttt ccctgcacct
      781 atttcaaaga tgttatcttt ttcatctaaa cttatgcaat tcattatttt ttctatgtga
      841 tattttgaag taataaaatt ttgactatct tttatattta ctttgttcat tataacctct
      901 ccttaattta ttgcatctct tttcgaatat ttatgttttt tgagaaaaga acgtactcat
      961 ggttcatccc gatatgcgta tcggtctgta tatcagcaac tttctatgtg tttcaactac
     1021 aatagtcatc tattctcatc tttctgagtc caccccctgc aaagcccctc tttacgacat
     1081 aaaaattcgg tcggaaaagg tatgcaaaag atgtttctct ctttaagaga aactcttcgg
     1141 gatgcaaaaa tatgaaaata actccaattc accaaattat atagcgactt ttttacaaaa
     1201 tgctaaaatt tgttgatttc cgtcaagcaa ttgttgagca aaaatgtctt ttacgataaa
     1261 atgatacctc aatatcaact gtttagcaaa acgatatttc tcttaaagag agaaacacct
     1321 ttttgttcac caatccccga cttttaatcc cgcggccatg attgaaaaag gaagagtatg
     1381 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1441 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1501 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1561 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1621 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1681 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1741 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1801 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1861 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1921 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1981 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2041 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtcacgc
     2101 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2161 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2221 ctgattaagc attggtaact gtcagaccaa gtttactcat aacgcgtcaa ttcgaggggg
     2281 atcaattccg tgataggtgg gctgcccttc ctggttggct tggtttcatc agccatccgc
     2341 ttgccctcat ctgttacgcc ggcggtagcc ggccagcctc gcagagcagg attcccgttg
     2401 agcaccgcca ggtgcgaata agggacagtg aagaaggaac acccgctcgc gggtgggcct
     2461 acttcaccta tcctgcccgg ctgacgccgt tggatacacc aaggaaagtc tacacgaacc
     2521 ctttggcaaa atcctgtata tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata
     2581 atgaccccga agcagggtta tgcagcggaa aacggaattg atccggccac gatgcgtccg
     2641 gcgtagagga tctgaagatc agcagttcaa cctgttgata gtacgtacta agctctcatg
     2701 tttcacgtac taagctctca tgtttaacgt actaagctct catgtttaac gaactaaacc
     2761 ctcatggcta acgtactaag ctctcatggc taacgtacta agctctcatg tttcacgtac
     2821 taagctctca tgtttgaaca ataaaattaa tataaatcag caacttaaat agcctctaag
     2881 gttttaagtt ttataagaaa aaaaagaata tataaggctt ttaaagcttt taaggtttaa
     2941 cggttgtgga caacaagcca gggatgtaac gcactgagaa gcccttagag cctctcaaag
     3001 caattttgag tgacacagga acacttaacg gctgacatgg gaattcccct ccaccgcggt
     3061 gtattaatgc ggctgcaatt tttttgggcg gggccgccca aaaaaatcct agcaccctgc
     3121 agggtctccc gctttttgca cccgctttcc aagagaagaa agccttgtta aattgactta
     3181 gtgtaaaagc gcagtactgc ttgaccataa gaacaaaaaa atctctatca ctgataggga
     3241 taaagtttgg aagataaagc taaaagttct tatctttgca gtctccctat cagtgataga
     3301 gacgcaagct gtcaccggat gtgctttccg gtctgatgag tccgtgagga cgaaacagcc
     3361 tctacaaata attttgttta acgcatttta aaataaaata aattatttat ttaattaaac
     3421 gaatatggtt tttactctgg aagattttgt tggcgattgg cgtcagaccg cgggttataa
     3481 tttggatcaa gtcctggaac agggtggcgt aagctctctg ttccagaacc tgggtgtgag
     3541 cgtgacgccg attcagcgca tcgttctgtc cggcgagaac ggtctgaaaa ttgatattca
     3601 tgtgatcatc ccgtacgaag gcctgagcgg tgaccaaatg ggtcaaatcg agaaaatctt
     3661 taaagtcgtc tacccagttg acgatcacca cttcaaggtt atcttgcatt acggtacgct
     3721 ggtgattgat ggtgtgaccc cgaatatgat tgactatttc ggccgtccgt atgaaggcat
     3781 tgccgttttt gacggtaaaa agatcaccgt caccggtacc ctgtggaatg gcaataagat
     3841 tattgacgag cgtctgatta acccggacgg cagcctgctg ttccgcgtga ccatcaacgg
     3901 tgtcacgggt tggcgtctgt gcgagcgcat cctggcataa caatgaatgg gctttcctcg
     3961 gtaccaaatt ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt ttggtcccta
     4021 attgcctatc ttccagtgat ggaacagcat ttgtgcattg gctgcaacaa tcagccttac
     4081 ttgtgcctgt tctatttccg aaccgaccgc ttgtatgaat ccatcaaaat tcgttttctc
     4141 tatgttggat tccttgttgc tcatattgtg atgataattt ctacaaatat agtcattggt
     4201 aactatctat gaaactgttt gatactttta ctcatcggat tcttttaata tcgctactct
     4261 ctacgttcaa ccaaaagaaa taatgactta ctgctatatt ttttgcacgt gtggggaaaa
     4321 tgtggggaaa attcaagcaa aagaaaaagc taagtattga actatcaaat acttagcttt
     4381 ctttcttgta cccagacccc gcatttgaaa taattaaagt ggggaaaatg tgggtaaaaa
     4441 gaaaaatgcg gaaaaacgcc acaattacac tgtatttcaa tatgttataa tcctattaaa
     4501 ttttaatcca agtttaatcg aattgcaaaa tatttagcag atgtggggaa aatgctgggg
     4561 aaaatattta tatttgcagc agagtaaaat tcaacctgaa agtctatgaa agtaaccttt
     4621 atcattaaaa aagcagccaa acgatatgat acagaatcca tggctacaat ctatgtccgt
     4681 tttagaaacg gaaggcagtt agactccgtt gctcctactc agttagccat caatcccaat
     4741 ctatgggatg ataaagacga atgtgtaaaa acgaaagctg tctgcaatga agaaatgcgt
     4801 acccatataa atgaagagat acgccagttg aaaacctata tcgagaaggt atatcaacaa
     4861 gaaaaggaag caatagacaa agaatggcta aaaacaacac ttgataaatt ttaccatcct
     4921 gaaaaatatt ttttgccgga ggaagtggtt atcaagccta ccattggaga actattcgat
     4981 gaatttctaa acaagcaccc tttgtcggaa gtacgaaaga aaaatttccg ggttgtcaaa
     5041 agagccttac tgcgttatga actatatgta agggctacaa agagaggaca aaagggcttt
     5101 atccttgatg tggatttggt aacacctgac acgcttcggg atatgtggga tttctttcag
     5161 aacgaatacc agtattatga actttacccg agcatttatg aagccattcc cgaaaagagg
     5221 acaccacagc ccagaagcaa aaacacgctg atagactgtt tttcaagaat acgcacattc
     5281 ttcctgtggt gcttcgataa caaacgcacc acaaacagac ctttcgacaa gtttccgata
     5341 gaggagtgta catatggtac accttattat ataacactcg aagaaaggga caggattttt
     5401 aatgcagacc tttctgccac cccacaactg gcaatacaga gggatatatt catatttcag
     5461 acactgatag gatgcagggt gagcgacctg taccgaatga ccaaactaaa tgtggtcaat
     5521 gaagccatag aatatattcc caagaaaacc aaagagggga atccggttac ggtacgtgtt
     5581 ccacttaacg acaaagcgaa agaaatcctt gaacgctaca aagaatatga gggaaaactg
     5641 ttgccgttca tatccgagca aaagtacaat gatgccataa aaaagatatt caaattagct
     5701 ggagttgacc gcatcgtaac aatcttagac ccgttgacgc acaacgaaat caaacgacct
     5761 atttatgaag tggcaagcag ccatctggca agacgtacgt ttatcggcaa tatctataaa
     5821 aaagtgaaag acccgaacct tgtttccgca ctgtcgggac acaaggaggg aagcaaagct
     5881 ttcagacgat acagggatat tgacgaagaa atgaagaaag accttgtaaa actattagac
     5941 tgataaaagt atcaaacagt ttcatagata gtt
//
