LOCUS       62056_17505        7200 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT451, complete sequence.
ACCESSION   MN991282
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7200)
  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 7200)
  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 7200)
  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..7200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41672.1"
                     /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-12"
                     /ncRNA_class="sgRNA"
     misc_RNA        3270..3345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3353..3411
                     /label=ECK120033736
                     /note="ECK120033736"
                     /regulatory_class="terminator"
     regulatory      3412..3419
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      3420..3503
                     /label=PBF2884
                     /note="PBF2884"
                     /regulatory_class="promoter"
     regulatory      3492..3495
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     misc_feature    3501..3502
                     /label=PAM
                     /note="PAM"
     ncRNA           3504..3523
                     /label=sgRNA
                     /note="sgRNA-12"
                     /ncRNA_class="sgRNA"
     misc_RNA        3529..3579
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3603..3645
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3646..4158
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      4169..4239
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      4247..4529
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      4490..4493
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4516..4523
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4530..4555
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="ribosome_binding_site"
     CDS             4556..5167
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41675.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      5184..5244
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     primer_bind     complement(5370..5390)
                     /label=pNBU1_seq.REV
                     /note="pNBU1_seq.REV"
     misc_recomb     5488..5817
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    5615..5628
                     /label=CC Region
                     /note="CC Region"
     misc_difference 5627
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             5833..7170
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41676.1"
                     /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 aaagatactt atgttagaag ttttagagct agaaatagca agttaaaata
     3301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttggatccaa
     3361 cgcatgagaa agcccccgga agatcacctt ccgggggctt ttttattgcg cgcggccgct
     3421 gctgtcatag gccggttgtc cggtagtccg gatgatttaa ggaagatttc tcttcttttt
     3481 tcactttctc cgaacaacct ccattctaac ataagtatct ttgcagtagc tgtcaccgga
     3541 tgtgctttcc ggtctgatga gtccgtgagg acgaaacagc ctctacaaat aattttgttt
     3601 aacgcatttt aaaataaaat aaattattta tttaattaaa cgaatatggt ttttactctg
     3661 gaagattttg ttggcgattg gcgtcagacc gcgggttata atttggatca agtcctggaa
     3721 cagggtggcg taagctctct gttccagaac ctgggtgtga gcgtgacgcc gattcagcgc
     3781 atcgttctgt ccggcgagaa cggtctgaaa attgatattc atgtgatcat cccgtacgaa
     3841 ggcctgagcg gtgaccaaat gggtcaaatc gagaaaatct ttaaagtcgt ctacccagtt
     3901 gacgatcacc acttcaaggt tatcttgcat tacggtacgc tggtgattga tggtgtgacc
     3961 ccgaatatga ttgactattt cggccgtccg tatgaaggca ttgccgtttt tgacggtaaa
     4021 aagatcaccg tcaccggtac cctgtggaat ggcaataaga ttattgacga gcgtctgatt
     4081 aacccggacg gcagcctgct gttccgcgtg accatcaacg gtgtcacggg ttggcgtctg
     4141 tgcgagcgca tcctggcata atgaactggg atccctcggt accaaatttt cgaaaaaaga
     4201 cgctgaaaag cgtctttttt cgttttggtc cgcggccgcc ggccgctgat ctggaagaag
     4261 caatgaaagc tgctgttaag tctccgaatc aggtattgtt cctgacaggt gtattcccat
     4321 ccggtaaacg cggatacttt gcagttgatc tgactcagga ataaattata aattaaggta
     4381 agaagattgt aggataagct aatgaaatag aaaaaggatg ccgtcacaca acttgtcggc
     4441 attctttttt gttttattag ttgaaaatat agtgaaaaag ttgcctaaat atgtatgtta
     4501 acaaattatt tgtcgtaact ttgcactcca aatctgtttt taacatatgg cactaatgca
     4561 gaaaaaattg actcgttctc agcagaaaca cttggacatc atcaacgctg ctaaagaaga
     4621 attcatcgaa ttcggtttct tggctgctaa catggaccgt atcacttctt ctgctgaagt
     4681 atctaaacgt actttgtacc gtcacttcga atctaaagaa gtattgttcg aatctgtatt
     4741 gactatcatc aacgactctg taaacgaatc tatctcttac cacttcgacc cgaacaaatc
     4801 tactgaagaa cagttgactg aaatcgctta caaagaaatc gacgtattgt acaaaactta
     4861 cggtatcgct ttggctcgta ctatcgtaat ggaattcttg cgtcagccgg aaatggctaa
     4921 aactttgatc cagaacatct actctatccg tgctatcact cagtggttcc gttctgctat
     4981 cgaagctaaa cgtttgaaag acgctgaccc gaaattgatg actgacgtat acgtatcttt
     5041 gttccagggt ttgttcttct ggccgcaggt aatgcacttg gacttggaac cgcacggtga
     5101 agaattgtct cagaaaatcg aaactttgac tactatcttc ttgcagtctt acggtgtagc
     5161 tgaataacaa tgaatgggct ttcctcggta ccaaattcca gaaaagaggc ctcccgaaag
     5221 gggggccttt tttcgttttg gtccctaatt gcctatcttc cagtgatgga acagcatttg
     5281 tgcattggct gcaacaatca gccttacttg tgcctgttct atttccgaac cgaccgcttg
     5341 tatgaatcca tcaaaattcg ttttctctat gttggattcc ttgttgctca tattgtgatg
     5401 ataatttcta caaatatagt cattggtaac tatctatgaa actgtttgat acttttactc
     5461 atcggattct tttaatatcg ctactctcta cgttcaacca aaagaaataa tgacttactg
     5521 ctatattttt tgcacgtgtg gggaaaatgt ggggaaaatt caagcaaaag aaaaagctaa
     5581 gtattgaact atcaaatact tagctttctt tcttgtaccc agaccccgca tttgaaataa
     5641 ttaaagtggg gaaaatgtgg gtaaaaagaa aaatgcggaa aaacgccaca attacactgt
     5701 atttcaatat gttataatcc tattaaattt taatccaagt ttaatcgaat tgcaaaatat
     5761 ttagcagatg tggggaaaat gctggggaaa atatttatat ttgcagcaga gtaaaattca
     5821 acctgaaagt ctatgaaagt aacctttatc attaaaaaag cagccaaacg atatgataca
     5881 gaatccatgg ctacaatcta tgtccgtttt agaaacggaa ggcagttaga ctccgttgct
     5941 cctactcagt tagccatcaa tcccaatcta tgggatgata aagacgaatg tgtaaaaacg
     6001 aaagctgtct gcaatgaaga aatgcgtacc catataaatg aagagatacg ccagttgaaa
     6061 acctatatcg agaaggtata tcaacaagaa aaggaagcaa tagacaaaga atggctaaaa
     6121 acaacacttg ataaatttta ccatcctgaa aaatattttt tgccggagga agtggttatc
     6181 aagcctacca ttggagaact attcgatgaa tttctaaaca agcacccttt gtcggaagta
     6241 cgaaagaaaa atttccgggt tgtcaaaaga gccttactgc gttatgaact atatgtaagg
     6301 gctacaaaga gaggacaaaa gggctttatc cttgatgtgg atttggtaac acctgacacg
     6361 cttcgggata tgtgggattt ctttcagaac gaataccagt attatgaact ttacccgagc
     6421 atttatgaag ccattcccga aaagaggaca ccacagccca gaagcaaaaa cacgctgata
     6481 gactgttttt caagaatacg cacattcttc ctgtggtgct tcgataacaa acgcaccaca
     6541 aacagacctt tcgacaagtt tccgatagag gagtgtacat atggtacacc ttattatata
     6601 acactcgaag aaagggacag gatttttaat gcagaccttt ctgccacccc acaactggca
     6661 atacagaggg atatattcat atttcagaca ctgataggat gcagggtgag cgacctgtac
     6721 cgaatgacca aactaaatgt ggtcaatgaa gccatagaat atattcccaa gaaaaccaaa
     6781 gaggggaatc cggttacggt acgtgttcca cttaacgaca aagcgaaaga aatccttgaa
     6841 cgctacaaag aatatgaggg aaaactgttg ccgttcatat ccgagcaaaa gtacaatgat
     6901 gccataaaaa agatattcaa attagctgga gttgaccgca tcgtaacaat cttagacccg
     6961 ttgacgcaca acgaaatcaa acgacctatt tatgaagtgg caagcagcca tctggcaaga
     7021 cgtacgttta tcggcaatat ctataaaaaa gtgaaagacc cgaaccttgt ttccgcactg
     7081 tcgggacaca aggagggaag caaagctttc agacgataca gggatattga cgaagaaatg
     7141 aagaaagacc ttgtaaaact attagactga taaaagtatc aaacagtttc atagatagtt
//
