LOCUS       62056_17500        7204 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT450, complete sequence.
ACCESSION   MN991281
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7204)
  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 7204)
  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 7204)
  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..7204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41667.1"
                     /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      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"
     regulatory      3213..3220
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     ncRNA           3250..3269
                     /label=sgRNA
                     /note="sgRNA-9"
                     /ncRNA_class="sgRNA"
     misc_RNA        3270..3345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3359..3415
                     /label=ECK120034435
                     /note="ECK120034435"
                     /regulatory_class="terminator"
     regulatory      3424..3507
                     /label=PBF2884
                     /note="PBF2884"
                     /regulatory_class="promoter"
     regulatory      3496..3499
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     misc_feature    3505..3506
                     /label=PAM
                     /note="PAM"
     ncRNA           3508..3527
                     /label=sgRNA
                     /note="sgRNA-9"
                     /ncRNA_class="sgRNA"
     misc_RNA        3533..3583
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3607..3649
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3650..4162
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      4173..4243
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      4251..4533
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      4494..4497
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4520..4527
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4534..4559
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="ribosome_binding_site"
     CDS             4560..5171
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41670.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      5188..5248
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     primer_bind     complement(5374..5394)
                     /label=pNBU1_seq.REV
                     /note="pNBU1_seq.REV"
     misc_recomb     5492..5821
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    5619..5632
                     /label=CC Region
                     /note="CC Region"
     misc_difference 5631
                     /replace="g"
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             5837..7174
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41671.1"
                     /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 gtttactttc aaagataatt aagttaaggg ttttagagct agaaatagca agttaaaata
     3301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttggatccct
     3361 cggtaccaaa ttccagaaaa gagacgctga aaagcgtctt ttttcgtttt ggtccgcggt
     3421 ggctgctgtc ataggccggt tgtccggtag tccggatgat ttaaggaaga tttctcttct
     3481 tttttcactt tctccgaaca acctccacct taacttaatt atctttgcag tagctgtcac
     3541 cggatgtgct ttccggtctg atgagtccgt gaggacgaaa cagcctctac aaataatttt
     3601 gtttaacgca ttttaaaata aaataaatta tttatttaat taaacgaata tggtttttac
     3661 tctggaagat tttgttggcg attggcgtca gaccgcgggt tataatttgg atcaagtcct
     3721 ggaacagggt ggcgtaagct ctctgttcca gaacctgggt gtgagcgtga cgccgattca
     3781 gcgcatcgtt ctgtccggcg agaacggtct gaaaattgat attcatgtga tcatcccgta
     3841 cgaaggcctg agcggtgacc aaatgggtca aatcgagaaa atctttaaag tcgtctaccc
     3901 agttgacgat caccacttca aggttatctt gcattacggt acgctggtga ttgatggtgt
     3961 gaccccgaat atgattgact atttcggccg tccgtatgaa ggcattgccg tttttgacgg
     4021 taaaaagatc accgtcaccg gtaccctgtg gaatggcaat aagattattg acgagcgtct
     4081 gattaacccg gacggcagcc tgctgttccg cgtgaccatc aacggtgtca cgggttggcg
     4141 tctgtgcgag cgcatcctgg cataatgaac tgggatccct cggtaccaaa ttttcgaaaa
     4201 aagacgctga aaagcgtctt ttttcgtttt ggtccgcggc cgccggccgc tgatctggaa
     4261 gaagcaatga aagctgctgt taagtctccg aatcaggtat tgttcctgac aggtgtattc
     4321 ccatccggta aacgcggata ctttgcagtt gatctgactc aggaataaat tataaattaa
     4381 ggtaagaaga ttgtaggata agctaatgaa atagaaaaag gatgccgtca cacaacttgt
     4441 cggcattctt ttttgtttta ttagttgaaa atatagtgaa aaagttgcct aaatatgtat
     4501 gttaacaaat tatttgtcgt aactttgcac tccaaatctg tttttaacat atggcactaa
     4561 tgcagaaaaa attgactcgt tctcagcaga aacacttgga catcatcaac gctgctaaag
     4621 aagaattcat cgaattcggt ttcttggctg ctaacatgga ccgtatcact tcttctgctg
     4681 aagtatctaa acgtactttg taccgtcact tcgaatctaa agaagtattg ttcgaatctg
     4741 tattgactat catcaacgac tctgtaaacg aatctatctc ttaccacttc gacccgaaca
     4801 aatctactga agaacagttg actgaaatcg cttacaaaga aatcgacgta ttgtacaaaa
     4861 cttacggtat cgctttggct cgtactatcg taatggaatt cttgcgtcag ccggaaatgg
     4921 ctaaaacttt gatccagaac atctactcta tccgtgctat cactcagtgg ttccgttctg
     4981 ctatcgaagc taaacgtttg aaagacgctg acccgaaatt gatgactgac gtatacgtat
     5041 ctttgttcca gggtttgttc ttctggccgc aggtaatgca cttggacttg gaaccgcacg
     5101 gtgaagaatt gtctcagaaa atcgaaactt tgactactat cttcttgcag tcttacggtg
     5161 tagctgaata acaatgaatg ggctttcctc ggtaccaaat tccagaaaag aggcctcccg
     5221 aaaggggggc cttttttcgt tttggtccct aattgcctat cttccagtga tggaacagca
     5281 tttgtgcatt ggctgcaaca atcagcctta cttgtgcctg ttctatttcc gaaccgaccg
     5341 cttgtatgaa tccatcaaaa ttcgttttct ctatgttgga ttccttgttg ctcatattgt
     5401 gatgataatt tctacaaata tagtcattgg taactatcta tgaaactgtt tgatactttt
     5461 actcatcgga ttcttttaat atcgctactc tctacgttca accaaaagaa ataatgactt
     5521 actgctatat tttttgcacg tgtggggaaa atgtggggaa aattcaagca aaagaaaaag
     5581 ctaagtattg aactatcaaa tacttagctt tctttcttgt acccagaccc cgcatttgaa
     5641 ataattaaag tggggaaaat gtgggtaaaa agaaaaatgc ggaaaaacgc cacaattaca
     5701 ctgtatttca atatgttata atcctattaa attttaatcc aagtttaatc gaattgcaaa
     5761 atatttagca gatgtgggga aaatgctggg gaaaatattt atatttgcag cagagtaaaa
     5821 ttcaacctga aagtctatga aagtaacctt tatcattaaa aaagcagcca aacgatatga
     5881 tacagaatcc atggctacaa tctatgtccg ttttagaaac ggaaggcagt tagactccgt
     5941 tgctcctact cagttagcca tcaatcccaa tctatgggat gataaagacg aatgtgtaaa
     6001 aacgaaagct gtctgcaatg aagaaatgcg tacccatata aatgaagaga tacgccagtt
     6061 gaaaacctat atcgagaagg tatatcaaca agaaaaggaa gcaatagaca aagaatggct
     6121 aaaaacaaca cttgataaat tttaccatcc tgaaaaatat tttttgccgg aggaagtggt
     6181 tatcaagcct accattggag aactattcga tgaatttcta aacaagcacc ctttgtcgga
     6241 agtacgaaag aaaaatttcc gggttgtcaa aagagcctta ctgcgttatg aactatatgt
     6301 aagggctaca aagagaggac aaaagggctt tatccttgat gtggatttgg taacacctga
     6361 cacgcttcgg gatatgtggg atttctttca gaacgaatac cagtattatg aactttaccc
     6421 gagcatttat gaagccattc ccgaaaagag gacaccacag cccagaagca aaaacacgct
     6481 gatagactgt ttttcaagaa tacgcacatt cttcctgtgg tgcttcgata acaaacgcac
     6541 cacaaacaga cctttcgaca agtttccgat agaggagtgt acatatggta caccttatta
     6601 tataacactc gaagaaaggg acaggatttt taatgcagac ctttctgcca ccccacaact
     6661 ggcaatacag agggatatat tcatatttca gacactgata ggatgcaggg tgagcgacct
     6721 gtaccgaatg accaaactaa atgtggtcaa tgaagccata gaatatattc ccaagaaaac
     6781 caaagagggg aatccggtta cggtacgtgt tccacttaac gacaaagcga aagaaatcct
     6841 tgaacgctac aaagaatatg agggaaaact gttgccgttc atatccgagc aaaagtacaa
     6901 tgatgccata aaaaagatat tcaaattagc tggagttgac cgcatcgtaa caatcttaga
     6961 cccgttgacg cacaacgaaa tcaaacgacc tatttatgaa gtggcaagca gccatctggc
     7021 aagacgtacg tttatcggca atatctataa aaaagtgaaa gacccgaacc ttgtttccgc
     7081 actgtcggga cacaaggagg gaagcaaagc tttcagacga tacagggata ttgacgaaga
     7141 aatgaagaaa gaccttgtaa aactattaga ctgataaaag tatcaaacag tttcatagat
     7201 agtt
//
