LOCUS       62056_17485        6922 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT447, complete sequence.
ACCESSION   MN991278
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6922)
  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 6922)
  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 6922)
  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..6922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41652.1"
                     /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"
     regulatory      3134..3176
                     /label=cfxA
                     /note="cfxA"
                     /regulatory_class="promoter"
     regulatory      3189..3192
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      3201..3212
                     /label=PcfxA
                     /note="PcfxA"
                     /regulatory_class="promoter"
     misc_RNA        3251..3301
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3325..3367
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3368..3880
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      3891..3961
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      3969..4251
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      4212..4215
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4238..4245
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4252..4277
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="ribosome_binding_site"
     CDS             4278..4889
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41656.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      4906..4966
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     primer_bind     complement(5092..5112)
                     /label=pNBU1_seq.REV
                     /note="pNBU1_seq.REV"
     misc_recomb     5210..5539
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    5337..5350
                     /label=CC Region
                     /note="CC Region"
     misc_difference 5349
                     /replace="g"
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             5555..6892
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41655.1"
                     /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 gctttacaaa gaaaattcga caaactgtta tttttctatc tatttaaatg
     3181 tacacccgtt tgtttacttt actttagtta tgtacctttg tcggcaatgt acacccgaaa
     3241 gtttacttta gctgtcaccg gatgtgcttt ccggtctgat gagtccgtga ggacgaaaca
     3301 gcctctacaa ataattttgt ttaacgcatt ttaaaataaa ataaattatt tatttaatta
     3361 aacgaatatg gtttttactc tggaagattt tgttggcgat tggcgtcaga ccgcgggtta
     3421 taatttggat caagtcctgg aacagggtgg cgtaagctct ctgttccaga acctgggtgt
     3481 gagcgtgacg ccgattcagc gcatcgttct gtccggcgag aacggtctga aaattgatat
     3541 tcatgtgatc atcccgtacg aaggcctgag cggtgaccaa atgggtcaaa tcgagaaaat
     3601 ctttaaagtc gtctacccag ttgacgatca ccacttcaag gttatcttgc attacggtac
     3661 gctggtgatt gatggtgtga ccccgaatat gattgactat ttcggccgtc cgtatgaagg
     3721 cattgccgtt tttgacggta aaaagatcac cgtcaccggt accctgtgga atggcaataa
     3781 gattattgac gagcgtctga ttaacccgga cggcagcctg ctgttccgcg tgaccatcaa
     3841 cggtgtcacg ggttggcgtc tgtgcgagcg catcctggca taatgaactg ggatccctcg
     3901 gtaccaaatt ttcgaaaaaa gacgctgaaa agcgtctttt ttcgttttgg tccgcggccg
     3961 ccggccgctg atctggaaga agcaatgaaa gctgctgtta agtctccgaa tcaggtattg
     4021 ttcctgacag gtgtattccc atccggtaaa cgcggatact ttgcagttga tctgactcag
     4081 gaataaatta taaattaagg taagaagatt gtaggataag ctaatgaaat agaaaaagga
     4141 tgccgtcaca caacttgtcg gcattctttt ttgttttatt agttgaaaat atagtgaaaa
     4201 agttgcctaa atatgtatgt taacaaatta tttgtcgtaa ctttgcactc caaatctgtt
     4261 tttaacatat ggcactaatg cagaaaaaat tgactcgttc tcagcagaaa cacttggaca
     4321 tcatcaacgc tgctaaagaa gaattcatcg aattcggttt cttggctgct aacatggacc
     4381 gtatcacttc ttctgctgaa gtatctaaac gtactttgta ccgtcacttc gaatctaaag
     4441 aagtattgtt cgaatctgta ttgactatca tcaacgactc tgtaaacgaa tctatctctt
     4501 accacttcga cccgaacaaa tctactgaag aacagttgac tgaaatcgct tacaaagaaa
     4561 tcgacgtatt gtacaaaact tacggtatcg ctttggctcg tactatcgta atggaattct
     4621 tgcgtcagcc ggaaatggct aaaactttga tccagaacat ctactctatc cgtgctatca
     4681 ctcagtggtt ccgttctgct atcgaagcta aacgtttgaa agacgctgac ccgaaattga
     4741 tgactgacgt atacgtatct ttgttccagg gtttgttctt ctggccgcag gtaatgcact
     4801 tggacttgga accgcacggt gaagaattgt ctcagaaaat cgaaactttg actactatct
     4861 tcttgcagtc ttacggtgta gctgaataac aatgaatggg ctttcctcgg taccaaattc
     4921 cagaaaagag gcctcccgaa aggggggcct tttttcgttt tggtccctaa ttgcctatct
     4981 tccagtgatg gaacagcatt tgtgcattgg ctgcaacaat cagccttact tgtgcctgtt
     5041 ctatttccga accgaccgct tgtatgaatc catcaaaatt cgttttctct atgttggatt
     5101 ccttgttgct catattgtga tgataatttc tacaaatata gtcattggta actatctatg
     5161 aaactgtttg atacttttac tcatcggatt cttttaatat cgctactctc tacgttcaac
     5221 caaaagaaat aatgacttac tgctatattt tttgcacgtg tggggaaaat gtggggaaaa
     5281 ttcaagcaaa agaaaaagct aagtattgaa ctatcaaata cttagctttc tttcttgtac
     5341 ccagaccccg catttgaaat aattaaagtg gggaaaatgt gggtaaaaag aaaaatgcgg
     5401 aaaaacgcca caattacact gtatttcaat atgttataat cctattaaat tttaatccaa
     5461 gtttaatcga attgcaaaat atttagcaga tgtggggaaa atgctgggga aaatatttat
     5521 atttgcagca gagtaaaatt caacctgaaa gtctatgaaa gtaaccttta tcattaaaaa
     5581 agcagccaaa cgatatgata cagaatccat ggctacaatc tatgtccgtt ttagaaacgg
     5641 aaggcagtta gactccgttg ctcctactca gttagccatc aatcccaatc tatgggatga
     5701 taaagacgaa tgtgtaaaaa cgaaagctgt ctgcaatgaa gaaatgcgta cccatataaa
     5761 tgaagagata cgccagttga aaacctatat cgagaaggta tatcaacaag aaaaggaagc
     5821 aatagacaaa gaatggctaa aaacaacact tgataaattt taccatcctg aaaaatattt
     5881 tttgccggag gaagtggtta tcaagcctac cattggagaa ctattcgatg aatttctaaa
     5941 caagcaccct ttgtcggaag tacgaaagaa aaatttccgg gttgtcaaaa gagccttact
     6001 gcgttatgaa ctatatgtaa gggctacaaa gagaggacaa aagggcttta tccttgatgt
     6061 ggatttggta acacctgaca cgcttcggga tatgtgggat ttctttcaga acgaatacca
     6121 gtattatgaa ctttacccga gcatttatga agccattccc gaaaagagga caccacagcc
     6181 cagaagcaaa aacacgctga tagactgttt ttcaagaata cgcacattct tcctgtggtg
     6241 cttcgataac aaacgcacca caaacagacc tttcgacaag tttccgatag aggagtgtac
     6301 atatggtaca ccttattata taacactcga agaaagggac aggattttta atgcagacct
     6361 ttctgccacc ccacaactgg caatacagag ggatatattc atatttcaga cactgatagg
     6421 atgcagggtg agcgacctgt accgaatgac caaactaaat gtggtcaatg aagccataga
     6481 atatattccc aagaaaacca aagaggggaa tccggttacg gtacgtgttc cacttaacga
     6541 caaagcgaaa gaaatccttg aacgctacaa agaatatgag ggaaaactgt tgccgttcat
     6601 atccgagcaa aagtacaatg atgccataaa aaagatattc aaattagctg gagttgaccg
     6661 catcgtaaca atcttagacc cgttgacgca caacgaaatc aaacgaccta tttatgaagt
     6721 ggcaagcagc catctggcaa gacgtacgtt tatcggcaat atctataaaa aagtgaaaga
     6781 cccgaacctt gtttccgcac tgtcgggaca caaggaggga agcaaagctt tcagacgata
     6841 cagggatatt gacgaagaaa tgaagaaaga ccttgtaaaa ctattagact gataaaagta
     6901 tcaaacagtt tcatagatag tt
//
