LOCUS       62056_17545        7196 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT494, complete sequence.
ACCESSION   MN991290
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7196)
  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 7196)
  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 7196)
  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..7196
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41711.1"
                     /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-14"
                     /ncRNA_class="sgRNA"
     misc_RNA        3270..3345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3353..3404
                     /label=pheA-1
                     /note="pheA-1"
     regulatory      3416..3491
                     /label=PBF2884
                     /note="PBF2884"
                     /regulatory_class="promoter"
     regulatory      3488..3491
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     misc_feature    3497..3498
                     /label=PAM
                     /note="PAM"
     ncRNA           3500..3519
                     /label=sgRNA
                     /note="sgRNA-14"
                     /ncRNA_class="sgRNA"
     misc_RNA        3525..3575
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3599..3641
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3642..4154
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      4165..4235
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      4243..4525
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      4486..4489
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     CDS             4552..5163
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41714.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      5180..5240
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     misc_recomb     5484..5813
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    5611..5624
                     /label=CC Region
                     /note="CC Region"
     misc_difference 5623
                     /label=mutation for higher integration efficiency
                     /note="mutation for higher integration efficiency"
     CDS             5829..7166
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41715.1"
                     /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 aaagatatat aactatatag ttttagagct agaaatagca agttaaaata
     3301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgacgaaca
     3361 ataaggcctc ccaaatcggg gggccttttt tattgataac aaaagccgcg gccgctgctg
     3421 tcataggccg gttgtccggt agtccggatg atttaaggaa gatttctctt cttttttcac
     3481 tttctccgaa caacctccat atatagttat atatctttgc agtagctgtc accggatgtg
     3541 ctttccggtc tgatgagtcc gtgaggacga aacagcctct acaaataatt ttgtttaacg
     3601 cattttaaaa taaaataaat tatttattta attaaacgaa tatggttttt actctggaag
     3661 attttgttgg cgattggcgt cagaccgcgg gttataattt ggatcaagtc ctggaacagg
     3721 gtggcgtaag ctctctgttc cagaacctgg gtgtgagcgt gacgccgatt cagcgcatcg
     3781 ttctgtccgg cgagaacggt ctgaaaattg atattcatgt gatcatcccg tacgaaggcc
     3841 tgagcggtga ccaaatgggt caaatcgaga aaatctttaa agtcgtctac ccagttgacg
     3901 atcaccactt caaggttatc ttgcattacg gtacgctggt gattgatggt gtgaccccga
     3961 atatgattga ctatttcggc cgtccgtatg aaggcattgc cgtttttgac ggtaaaaaga
     4021 tcaccgtcac cggtaccctg tggaatggca ataagattat tgacgagcgt ctgattaacc
     4081 cggacggcag cctgctgttc cgcgtgacca tcaacggtgt cacgggttgg cgtctgtgcg
     4141 agcgcatcct ggcataatga actgggatcc ctcggtacca aattttcgaa aaaagacgct
     4201 gaaaagcgtc ttttttcgtt ttggtccgcg gccgccggcc gctgatctgg aagaagcaat
     4261 gaaagctgct gttaagtctc cgaatcaggt attgttcctg acaggtgtat tcccatccgg
     4321 taaacgcgga tactttgcag ttgatctgac tcaggaataa attataaatt aaggtaagaa
     4381 gattgtagga taagctaatg aaatagaaaa aggatgccgt cacacaactt gtcggcattc
     4441 ttttttgttt tattagttga aaatatagtg aaaaagttgc ctaaatatgt atgttaacaa
     4501 attatttgtc gtaactttgc actccaaatc tgtttttaac atatggcact aatgcagaaa
     4561 aaattgactc gttctcagca gaaacacttg gacatcatca acgctgctaa agaagaattc
     4621 atcgaattcg gtttcttggc tgctaacatg gaccgtatca cttcttctgc tgaagtatct
     4681 aaacgtactt tgtaccgtca cttcgaatct aaagaagtat tgttcgaatc tgtattgact
     4741 atcatcaacg actctgtaaa cgaatctatc tcttaccact tcgacccgaa caaatctact
     4801 gaagaacagt tgactgaaat cgcttacaaa gaaatcgacg tattgtacaa aacttacggt
     4861 atcgctttgg ctcgtactat cgtaatggaa ttcttgcgtc agccggaaat ggctaaaact
     4921 ttgatccaga acatctactc tatccgtgct atcactcagt ggttccgttc tgctatcgaa
     4981 gctaaacgtt tgaaagacgc tgacccgaaa ttgatgactg acgtatacgt atctttgttc
     5041 cagggtttgt tcttctggcc gcaggtaatg cacttggact tggaaccgca cggtgaagaa
     5101 ttgtctcaga aaatcgaaac tttgactact atcttcttgc agtcttacgg tgtagctgaa
     5161 taacaatgaa tgggctttcc tcggtaccaa attccagaaa agaggcctcc cgaaaggggg
     5221 gccttttttc gttttggtcc ctaattgcct atcttccagt gatggaacag catttgtgca
     5281 ttggctgcaa caatcagcct tacttgtgcc tgttctattt ccgaaccgac cgcttgtatg
     5341 aatccatcaa aattcgtttt ctctatgttg gattccttgt tgctcatatt gtgatgataa
     5401 tttctacaaa tatagtcatt ggtaactatc tatgaaactg tttgatactt ttactcatcg
     5461 gattctttta atatcgctac tctctacgtt caaccaaaag aaataatgac ttactgctat
     5521 attttttgca cgtgtgggga aaatgtgggg aaaattcaag caaaagaaaa agctaagtat
     5581 tgaactatca aatacttagc tttctttctt gtacccagac cccgcatttg aaataattaa
     5641 agtggggaaa atgtgggtaa aaagaaaaat gcggaaaaac gccacaatta cactgtattt
     5701 caatatgtta taatcctatt aaattttaat ccaagtttaa tcgaattgca aaatatttag
     5761 cagatgtggg gaaaatgctg gggaaaatat ttatatttgc agcagagtaa aattcaacct
     5821 gaaagtctat gaaagtaacc tttatcatta aaaaagcagc caaacgatat gatacagaat
     5881 ccatggctac aatctatgtc cgttttagaa acggaaggca gttagactcc gttgctccta
     5941 ctcagttagc catcaatccc aatctatggg atgataaaga cgaatgtgta aaaacgaaag
     6001 ctgtctgcaa tgaagaaatg cgtacccata taaatgaaga gatacgccag ttgaaaacct
     6061 atatcgagaa ggtatatcaa caagaaaagg aagcaataga caaagaatgg ctaaaaacaa
     6121 cacttgataa attttaccat cctgaaaaat attttttgcc ggaggaagtg gttatcaagc
     6181 ctaccattgg agaactattc gatgaatttc taaacaagca ccctttgtcg gaagtacgaa
     6241 agaaaaattt ccgggttgtc aaaagagcct tactgcgtta tgaactatat gtaagggcta
     6301 caaagagagg acaaaagggc tttatccttg atgtggattt ggtaacacct gacacgcttc
     6361 gggatatgtg ggatttcttt cagaacgaat accagtatta tgaactttac ccgagcattt
     6421 atgaagccat tcccgaaaag aggacaccac agcccagaag caaaaacacg ctgatagact
     6481 gtttttcaag aatacgcaca ttcttcctgt ggtgcttcga taacaaacgc accacaaaca
     6541 gacctttcga caagtttccg atagaggagt gtacatatgg tacaccttat tatataacac
     6601 tcgaagaaag ggacaggatt tttaatgcag acctttctgc caccccacaa ctggcaatac
     6661 agagggatat attcatattt cagacactga taggatgcag ggtgagcgac ctgtaccgaa
     6721 tgaccaaact aaatgtggtc aatgaagcca tagaatatat tcccaagaaa accaaagagg
     6781 ggaatccggt tacggtacgt gttccactta acgacaaagc gaaagaaatc cttgaacgct
     6841 acaaagaata tgagggaaaa ctgttgccgt tcatatccga gcaaaagtac aatgatgcca
     6901 taaaaaagat attcaaatta gctggagttg accgcatcgt aacaatctta gacccgttga
     6961 cgcacaacga aatcaaacga cctatttatg aagtggcaag cagccatctg gcaagacgta
     7021 cgtttatcgg caatatctat aaaaaagtga aagacccgaa ccttgtttcc gcactgtcgg
     7081 gacacaagga gggaagcaaa gctttcagac gatacaggga tattgacgaa gaaatgaaga
     7141 aagaccttgt aaaactatta gactgataaa agtatcaaac agtttcatag atagtt
//
