LOCUS       62056_17490        7194 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT448, complete sequence.
ACCESSION   MN991279
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7194)
  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 7194)
  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 7194)
  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..7194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41657.1"
                     /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"
     misc_feature    3123..3129
                     /label=Scar
                     /note="Scar"
     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"
     misc_feature    3201..3212
                     /label=from PcfxA
                     /note="from PcfxA"
     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-7"
                     /ncRNA_class="sgRNA"
     misc_RNA        3270..3345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3353..3402
                     /label=L3S3P41
                     /note="L3S3P41"
                     /regulatory_class="terminator"
     regulatory      3414..3489
                     /label=PBF2884
                     /note="PBF2884"
                     /regulatory_class="promoter"
     regulatory      3486..3489
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     misc_feature    3495..3496
                     /label=PAM
                     /note="PAM"
     ncRNA           3498..3517
                     /label=sgRNA
                     /note="sgRNA-7"
                     /ncRNA_class="sgRNA"
     misc_RNA        3523..3573
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3597..3639
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3640..4152
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      4163..4233
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      4241..4523
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      4484..4487
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4510..4517
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4524..4549
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="ribosome_binding_site"
     CDS             4550..5161
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41661.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      5178..5238
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     primer_bind     complement(5364..5384)
                     /label=pNBU1_seq.REV
                     /note="pNBU1_seq.REV"
     misc_recomb     5482..5811
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    5609..5622
                     /label=CC Region
                     /note="CC Region"
     misc_difference 5621
                     /replace="g"
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             5827..7164
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41660.1"
                     /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 aaagataata gaacttaagg ttttagagct agaaatagca agttaaaata
     3301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttggatccaa
     3361 aaaaaaaaaa caccctaacg ggtgtttttt tttttttggt ctgccgcggc cgctgctgtc
     3421 ataggccggt tgtccggtag tccggatgat ttaaggaaga tttctcttct tttttcactt
     3481 tctccgaaca acctccactt aagttctatt atctttgcag tagctgtcac cggatgtgct
     3541 ttccggtctg atgagtccgt gaggacgaaa cagcctctac aaataatttt gtttaacgca
     3601 ttttaaaata aaataaatta tttatttaat taaacgaata tggtttttac tctggaagat
     3661 tttgttggcg attggcgtca gaccgcgggt tataatttgg atcaagtcct ggaacagggt
     3721 ggcgtaagct ctctgttcca gaacctgggt gtgagcgtga cgccgattca gcgcatcgtt
     3781 ctgtccggcg agaacggtct gaaaattgat attcatgtga tcatcccgta cgaaggcctg
     3841 agcggtgacc aaatgggtca aatcgagaaa atctttaaag tcgtctaccc agttgacgat
     3901 caccacttca aggttatctt gcattacggt acgctggtga ttgatggtgt gaccccgaat
     3961 atgattgact atttcggccg tccgtatgaa ggcattgccg tttttgacgg taaaaagatc
     4021 accgtcaccg gtaccctgtg gaatggcaat aagattattg acgagcgtct gattaacccg
     4081 gacggcagcc tgctgttccg cgtgaccatc aacggtgtca cgggttggcg tctgtgcgag
     4141 cgcatcctgg cataatgaac tgggatccct cggtaccaaa ttttcgaaaa aagacgctga
     4201 aaagcgtctt ttttcgtttt ggtccgcggc cgccggccgc tgatctggaa gaagcaatga
     4261 aagctgctgt taagtctccg aatcaggtat tgttcctgac aggtgtattc ccatccggta
     4321 aacgcggata ctttgcagtt gatctgactc aggaataaat tataaattaa ggtaagaaga
     4381 ttgtaggata agctaatgaa atagaaaaag gatgccgtca cacaacttgt cggcattctt
     4441 ttttgtttta ttagttgaaa atatagtgaa aaagttgcct aaatatgtat gttaacaaat
     4501 tatttgtcgt aactttgcac tccaaatctg tttttaacat atggcactaa tgcagaaaaa
     4561 attgactcgt tctcagcaga aacacttgga catcatcaac gctgctaaag aagaattcat
     4621 cgaattcggt ttcttggctg ctaacatgga ccgtatcact tcttctgctg aagtatctaa
     4681 acgtactttg taccgtcact tcgaatctaa agaagtattg ttcgaatctg tattgactat
     4741 catcaacgac tctgtaaacg aatctatctc ttaccacttc gacccgaaca aatctactga
     4801 agaacagttg actgaaatcg cttacaaaga aatcgacgta ttgtacaaaa cttacggtat
     4861 cgctttggct cgtactatcg taatggaatt cttgcgtcag ccggaaatgg ctaaaacttt
     4921 gatccagaac atctactcta tccgtgctat cactcagtgg ttccgttctg ctatcgaagc
     4981 taaacgtttg aaagacgctg acccgaaatt gatgactgac gtatacgtat ctttgttcca
     5041 gggtttgttc ttctggccgc aggtaatgca cttggacttg gaaccgcacg gtgaagaatt
     5101 gtctcagaaa atcgaaactt tgactactat cttcttgcag tcttacggtg tagctgaata
     5161 acaatgaatg ggctttcctc ggtaccaaat tccagaaaag aggcctcccg aaaggggggc
     5221 cttttttcgt tttggtccct aattgcctat cttccagtga tggaacagca tttgtgcatt
     5281 ggctgcaaca atcagcctta cttgtgcctg ttctatttcc gaaccgaccg cttgtatgaa
     5341 tccatcaaaa ttcgttttct ctatgttgga ttccttgttg ctcatattgt gatgataatt
     5401 tctacaaata tagtcattgg taactatcta tgaaactgtt tgatactttt actcatcgga
     5461 ttcttttaat atcgctactc tctacgttca accaaaagaa ataatgactt actgctatat
     5521 tttttgcacg tgtggggaaa atgtggggaa aattcaagca aaagaaaaag ctaagtattg
     5581 aactatcaaa tacttagctt tctttcttgt acccagaccc cgcatttgaa ataattaaag
     5641 tggggaaaat gtgggtaaaa agaaaaatgc ggaaaaacgc cacaattaca ctgtatttca
     5701 atatgttata atcctattaa attttaatcc aagtttaatc gaattgcaaa atatttagca
     5761 gatgtgggga aaatgctggg gaaaatattt atatttgcag cagagtaaaa ttcaacctga
     5821 aagtctatga aagtaacctt tatcattaaa aaagcagcca aacgatatga tacagaatcc
     5881 atggctacaa tctatgtccg ttttagaaac ggaaggcagt tagactccgt tgctcctact
     5941 cagttagcca tcaatcccaa tctatgggat gataaagacg aatgtgtaaa aacgaaagct
     6001 gtctgcaatg aagaaatgcg tacccatata aatgaagaga tacgccagtt gaaaacctat
     6061 atcgagaagg tatatcaaca agaaaaggaa gcaatagaca aagaatggct aaaaacaaca
     6121 cttgataaat tttaccatcc tgaaaaatat tttttgccgg aggaagtggt tatcaagcct
     6181 accattggag aactattcga tgaatttcta aacaagcacc ctttgtcgga agtacgaaag
     6241 aaaaatttcc gggttgtcaa aagagcctta ctgcgttatg aactatatgt aagggctaca
     6301 aagagaggac aaaagggctt tatccttgat gtggatttgg taacacctga cacgcttcgg
     6361 gatatgtggg atttctttca gaacgaatac cagtattatg aactttaccc gagcatttat
     6421 gaagccattc ccgaaaagag gacaccacag cccagaagca aaaacacgct gatagactgt
     6481 ttttcaagaa tacgcacatt cttcctgtgg tgcttcgata acaaacgcac cacaaacaga
     6541 cctttcgaca agtttccgat agaggagtgt acatatggta caccttatta tataacactc
     6601 gaagaaaggg acaggatttt taatgcagac ctttctgcca ccccacaact ggcaatacag
     6661 agggatatat tcatatttca gacactgata ggatgcaggg tgagcgacct gtaccgaatg
     6721 accaaactaa atgtggtcaa tgaagccata gaatatattc ccaagaaaac caaagagggg
     6781 aatccggtta cggtacgtgt tccacttaac gacaaagcga aagaaatcct tgaacgctac
     6841 aaagaatatg agggaaaact gttgccgttc atatccgagc aaaagtacaa tgatgccata
     6901 aaaaagatat tcaaattagc tggagttgac cgcatcgtaa caatcttaga cccgttgacg
     6961 cacaacgaaa tcaaacgacc tatttatgaa gtggcaagca gccatctggc aagacgtacg
     7021 tttatcggca atatctataa aaaagtgaaa gacccgaacc ttgtttccgc actgtcggga
     7081 cacaaggagg gaagcaaagc tttcagacga tacagggata ttgacgaaga aatgaagaaa
     7141 gaccttgtaa aactattaga ctgataaaag tatcaaacag tttcatagat agtt
//
