LOCUS       62056_17495        7237 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT449, complete sequence.
ACCESSION   MN991280
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7237)
  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 7237)
  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 7237)
  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..7237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41662.1"
                     /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"
     misc_difference 3062
                     /replace="g"
     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-8"
                     /ncRNA_class="sgRNA"
     misc_RNA        3270..3345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3353..3448
                     /label=ECK120029600
                     /note="ECK120029600"
                     /regulatory_class="terminator"
     regulatory      3449..3456
                     /label=ECK120029600
                     /note="ECK120029600"
                     /regulatory_class="terminator"
     regulatory      3457..3540
                     /label=PBF2884
                     /note="PBF2884"
                     /regulatory_class="promoter"
     regulatory      3529..3532
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     misc_feature    3538..3539
                     /label=PAM
                     /note="PAM"
     ncRNA           3541..3560
                     /label=sgRNA
                     /note="sgRNA-8"
                     /ncRNA_class="sgRNA"
     misc_RNA        3566..3616
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3640..3682
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3683..4195
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      4206..4276
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      4284..4566
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      4527..4530
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4553..4560
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4567..4592
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="ribosome_binding_site"
     CDS             4593..5204
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41665.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      5221..5281
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     misc_recomb     5525..5854
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    5652..5665
                     /label=CC Region
                     /note="CC Region"
     misc_difference 5664
                     /replace="g"
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             5870..7207
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41666.1"
                     /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 aaagataaat cagagaaaag ttttagagct agaaatagca agttaaaata
     3301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttggatcctt
     3361 cagtcaaaaa acttaagacc gccggtcttg tccactacct tgcagtaatg cggtggacag
     3421 gatcggcggt tttcttttct cttctcaagc ggccgctgct gtcataggcc ggttgtccgg
     3481 tagtccggat gatttaagga agatttctct tcttttttca ctttctccga acaacctcca
     3541 ttttctctga tttatctttg cagtagctgt caccggatgt gctttccggt ctgatgagtc
     3601 cgtgaggacg aaacagcctc tacaaataat tttgtttaac gcattttaaa ataaaataaa
     3661 ttatttattt aattaaacga atatggtttt tactctggaa gattttgttg gcgattggcg
     3721 tcagaccgcg ggttataatt tggatcaagt cctggaacag ggtggcgtaa gctctctgtt
     3781 ccagaacctg ggtgtgagcg tgacgccgat tcagcgcatc gttctgtccg gcgagaacgg
     3841 tctgaaaatt gatattcatg tgatcatccc gtacgaaggc ctgagcggtg accaaatggg
     3901 tcaaatcgag aaaatcttta aagtcgtcta cccagttgac gatcaccact tcaaggttat
     3961 cttgcattac ggtacgctgg tgattgatgg tgtgaccccg aatatgattg actatttcgg
     4021 ccgtccgtat gaaggcattg ccgtttttga cggtaaaaag atcaccgtca ccggtaccct
     4081 gtggaatggc aataagatta ttgacgagcg tctgattaac ccggacggca gcctgctgtt
     4141 ccgcgtgacc atcaacggtg tcacgggttg gcgtctgtgc gagcgcatcc tggcataatg
     4201 aactgggatc cctcggtacc aaattttcga aaaaagacgc tgaaaagcgt cttttttcgt
     4261 tttggtccgc ggccgccggc cgctgatctg gaagaagcaa tgaaagctgc tgttaagtct
     4321 ccgaatcagg tattgttcct gacaggtgta ttcccatccg gtaaacgcgg atactttgca
     4381 gttgatctga ctcaggaata aattataaat taaggtaaga agattgtagg ataagctaat
     4441 gaaatagaaa aaggatgccg tcacacaact tgtcggcatt cttttttgtt ttattagttg
     4501 aaaatatagt gaaaaagttg cctaaatatg tatgttaaca aattatttgt cgtaactttg
     4561 cactccaaat ctgtttttaa catatggcac taatgcagaa aaaattgact cgttctcagc
     4621 agaaacactt ggacatcatc aacgctgcta aagaagaatt catcgaattc ggtttcttgg
     4681 ctgctaacat ggaccgtatc acttcttctg ctgaagtatc taaacgtact ttgtaccgtc
     4741 acttcgaatc taaagaagta ttgttcgaat ctgtattgac tatcatcaac gactctgtaa
     4801 acgaatctat ctcttaccac ttcgacccga acaaatctac tgaagaacag ttgactgaaa
     4861 tcgcttacaa agaaatcgac gtattgtaca aaacttacgg tatcgctttg gctcgtacta
     4921 tcgtaatgga attcttgcgt cagccggaaa tggctaaaac tttgatccag aacatctact
     4981 ctatccgtgc tatcactcag tggttccgtt ctgctatcga agctaaacgt ttgaaagacg
     5041 ctgacccgaa attgatgact gacgtatacg tatctttgtt ccagggtttg ttcttctggc
     5101 cgcaggtaat gcacttggac ttggaaccgc acggtgaaga attgtctcag aaaatcgaaa
     5161 ctttgactac tatcttcttg cagtcttacg gtgtagctga ataacaatga atgggctttc
     5221 ctcggtacca aattccagaa aagaggcctc ccgaaagggg ggcctttttt cgttttggtc
     5281 cctaattgcc tatcttccag tgatggaaca gcatttgtgc attggctgca acaatcagcc
     5341 ttacttgtgc ctgttctatt tccgaaccga ccgcttgtat gaatccatca aaattcgttt
     5401 tctctatgtt ggattccttg ttgctcatat tgtgatgata atttctacaa atatagtcat
     5461 tggtaactat ctatgaaact gtttgatact tttactcatc ggattctttt aatatcgcta
     5521 ctctctacgt tcaaccaaaa gaaataatga cttactgcta tattttttgc acgtgtgggg
     5581 aaaatgtggg gaaaattcaa gcaaaagaaa aagctaagta ttgaactatc aaatacttag
     5641 ctttctttct tgtacccaga ccccgcattt gaaataatta aagtggggaa aatgtgggta
     5701 aaaagaaaaa tgcggaaaaa cgccacaatt acactgtatt tcaatatgtt ataatcctat
     5761 taaattttaa tccaagttta atcgaattgc aaaatattta gcagatgtgg ggaaaatgct
     5821 ggggaaaata tttatatttg cagcagagta aaattcaacc tgaaagtcta tgaaagtaac
     5881 ctttatcatt aaaaaagcag ccaaacgata tgatacagaa tccatggcta caatctatgt
     5941 ccgttttaga aacggaaggc agttagactc cgttgctcct actcagttag ccatcaatcc
     6001 caatctatgg gatgataaag acgaatgtgt aaaaacgaaa gctgtctgca atgaagaaat
     6061 gcgtacccat ataaatgaag agatacgcca gttgaaaacc tatatcgaga aggtatatca
     6121 acaagaaaag gaagcaatag acaaagaatg gctaaaaaca acacttgata aattttacca
     6181 tcctgaaaaa tattttttgc cggaggaagt ggttatcaag cctaccattg gagaactatt
     6241 cgatgaattt ctaaacaagc accctttgtc ggaagtacga aagaaaaatt tccgggttgt
     6301 caaaagagcc ttactgcgtt atgaactata tgtaagggct acaaagagag gacaaaaggg
     6361 ctttatcctt gatgtggatt tggtaacacc tgacacgctt cgggatatgt gggatttctt
     6421 tcagaacgaa taccagtatt atgaacttta cccgagcatt tatgaagcca ttcccgaaaa
     6481 gaggacacca cagcccagaa gcaaaaacac gctgatagac tgtttttcaa gaatacgcac
     6541 attcttcctg tggtgcttcg ataacaaacg caccacaaac agacctttcg acaagtttcc
     6601 gatagaggag tgtacatatg gtacacctta ttatataaca ctcgaagaaa gggacaggat
     6661 ttttaatgca gacctttctg ccaccccaca actggcaata cagagggata tattcatatt
     6721 tcagacactg ataggatgca gggtgagcga cctgtaccga atgaccaaac taaatgtggt
     6781 caatgaagcc atagaatata ttcccaagaa aaccaaagag gggaatccgg ttacggtacg
     6841 tgttccactt aacgacaaag cgaaagaaat ccttgaacgc tacaaagaat atgagggaaa
     6901 actgttgccg ttcatatccg agcaaaagta caatgatgcc ataaaaaaga tattcaaatt
     6961 agctggagtt gaccgcatcg taacaatctt agacccgttg acgcacaacg aaatcaaacg
     7021 acctatttat gaagtggcaa gcagccatct ggcaagacgt acgtttatcg gcaatatcta
     7081 taaaaaagtg aaagacccga accttgtttc cgcactgtcg ggacacaagg agggaagcaa
     7141 agctttcaga cgatacaggg atattgacga agaaatgaag aaagaccttg taaaactatt
     7201 agactgataa aagtatcaaa cagtttcata gatagtt
//
