LOCUS       62056_17535        7191 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT492, complete sequence.
ACCESSION   MN991288
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7191)
  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 7191)
  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 7191)
  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..7191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41701.1"
                     /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-10"
                     /ncRNA_class="sgRNA"
     misc_RNA        3270..3345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3353..3399
                     /label=ECK125109870
                     /note="ECK125109870"
                     /regulatory_class="terminator"
     regulatory      3411..3486
                     /label=PBF2884
                     /note="PBF2884"
                     /regulatory_class="promoter"
     regulatory      3483..3486
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     misc_feature    3492..3493
                     /label=PAM
                     /note="PAM"
     ncRNA           3495..3514
                     /label=sgRNA
                     /note="sgRNA-10"
                     /ncRNA_class="sgRNA"
     misc_RNA        3520..3570
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3594..3636
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3637..4149
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      4160..4230
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      4238..4520
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      4481..4484
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4507..4514
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     CDS             4547..5158
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41704.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      5175..5235
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     primer_bind     complement(5361..5381)
                     /label=pNBU1_seq.REV
                     /note="pNBU1_seq.REV"
     misc_recomb     5479..5808
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    5606..5619
                     /label=CC Region
                     /note="CC Region"
     misc_difference 5618
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             5824..7161
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41705.1"
                     /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 aaagatagaa taagtctttg ttttagagct agaaatagca agttaaaata
     3301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttccaattat
     3361 tgaacaccct aacgggtgtt tttttgtttc tggtctcccg ccgcggccgc tgctgtcata
     3421 ggccggttgt ccggtagtcc ggatgattta aggaagattt ctcttctttt ttcactttct
     3481 ccgaacaacc tccaaaagac ttattctatc tttgcagtag ctgtcaccgg atgtgctttc
     3541 cggtctgatg agtccgtgag gacgaaacag cctctacaaa taattttgtt taacgcattt
     3601 taaaataaaa taaattattt atttaattaa acgaatatgg tttttactct ggaagatttt
     3661 gttggcgatt ggcgtcagac cgcgggttat aatttggatc aagtcctgga acagggtggc
     3721 gtaagctctc tgttccagaa cctgggtgtg agcgtgacgc cgattcagcg catcgttctg
     3781 tccggcgaga acggtctgaa aattgatatt catgtgatca tcccgtacga aggcctgagc
     3841 ggtgaccaaa tgggtcaaat cgagaaaatc tttaaagtcg tctacccagt tgacgatcac
     3901 cacttcaagg ttatcttgca ttacggtacg ctggtgattg atggtgtgac cccgaatatg
     3961 attgactatt tcggccgtcc gtatgaaggc attgccgttt ttgacggtaa aaagatcacc
     4021 gtcaccggta ccctgtggaa tggcaataag attattgacg agcgtctgat taacccggac
     4081 ggcagcctgc tgttccgcgt gaccatcaac ggtgtcacgg gttggcgtct gtgcgagcgc
     4141 atcctggcat aatgaactgg gatccctcgg taccaaattt tcgaaaaaag acgctgaaaa
     4201 gcgtcttttt tcgttttggt ccgcggccgc cggccgctga tctggaagaa gcaatgaaag
     4261 ctgctgttaa gtctccgaat caggtattgt tcctgacagg tgtattccca tccggtaaac
     4321 gcggatactt tgcagttgat ctgactcagg aataaattat aaattaaggt aagaagattg
     4381 taggataagc taatgaaata gaaaaaggat gccgtcacac aacttgtcgg cattcttttt
     4441 tgttttatta gttgaaaata tagtgaaaaa gttgcctaaa tatgtatgtt aacaaattat
     4501 ttgtcgtaac tttgcactcc aaatctgttt ttaacatatg gcactaatgc agaaaaaatt
     4561 gactcgttct cagcagaaac acttggacat catcaacgct gctaaagaag aattcatcga
     4621 attcggtttc ttggctgcta acatggaccg tatcacttct tctgctgaag tatctaaacg
     4681 tactttgtac cgtcacttcg aatctaaaga agtattgttc gaatctgtat tgactatcat
     4741 caacgactct gtaaacgaat ctatctctta ccacttcgac ccgaacaaat ctactgaaga
     4801 acagttgact gaaatcgctt acaaagaaat cgacgtattg tacaaaactt acggtatcgc
     4861 tttggctcgt actatcgtaa tggaattctt gcgtcagccg gaaatggcta aaactttgat
     4921 ccagaacatc tactctatcc gtgctatcac tcagtggttc cgttctgcta tcgaagctaa
     4981 acgtttgaaa gacgctgacc cgaaattgat gactgacgta tacgtatctt tgttccaggg
     5041 tttgttcttc tggccgcagg taatgcactt ggacttggaa ccgcacggtg aagaattgtc
     5101 tcagaaaatc gaaactttga ctactatctt cttgcagtct tacggtgtag ctgaataaca
     5161 atgaatgggc tttcctcggt accaaattcc agaaaagagg cctcccgaaa ggggggcctt
     5221 ttttcgtttt ggtccctaat tgcctatctt ccagtgatgg aacagcattt gtgcattggc
     5281 tgcaacaatc agccttactt gtgcctgttc tatttccgaa ccgaccgctt gtatgaatcc
     5341 atcaaaattc gttttctcta tgttggattc cttgttgctc atattgtgat gataatttct
     5401 acaaatatag tcattggtaa ctatctatga aactgtttga tacttttact catcggattc
     5461 ttttaatatc gctactctct acgttcaacc aaaagaaata atgacttact gctatatttt
     5521 ttgcacgtgt ggggaaaatg tggggaaaat tcaagcaaaa gaaaaagcta agtattgaac
     5581 tatcaaatac ttagctttct ttcttgtacc cagaccccgc atttgaaata attaaagtgg
     5641 ggaaaatgtg ggtaaaaaga aaaatgcgga aaaacgccac aattacactg tatttcaata
     5701 tgttataatc ctattaaatt ttaatccaag tttaatcgaa ttgcaaaata tttagcagat
     5761 gtggggaaaa tgctggggaa aatatttata tttgcagcag agtaaaattc aacctgaaag
     5821 tctatgaaag taacctttat cattaaaaaa gcagccaaac gatatgatac agaatccatg
     5881 gctacaatct atgtccgttt tagaaacgga aggcagttag actccgttgc tcctactcag
     5941 ttagccatca atcccaatct atgggatgat aaagacgaat gtgtaaaaac gaaagctgtc
     6001 tgcaatgaag aaatgcgtac ccatataaat gaagagatac gccagttgaa aacctatatc
     6061 gagaaggtat atcaacaaga aaaggaagca atagacaaag aatggctaaa aacaacactt
     6121 gataaatttt accatcctga aaaatatttt ttgccggagg aagtggttat caagcctacc
     6181 attggagaac tattcgatga atttctaaac aagcaccctt tgtcggaagt acgaaagaaa
     6241 aatttccggg ttgtcaaaag agccttactg cgttatgaac tatatgtaag ggctacaaag
     6301 agaggacaaa agggctttat ccttgatgtg gatttggtaa cacctgacac gcttcgggat
     6361 atgtgggatt tctttcagaa cgaataccag tattatgaac tttacccgag catttatgaa
     6421 gccattcccg aaaagaggac accacagccc agaagcaaaa acacgctgat agactgtttt
     6481 tcaagaatac gcacattctt cctgtggtgc ttcgataaca aacgcaccac aaacagacct
     6541 ttcgacaagt ttccgataga ggagtgtaca tatggtacac cttattatat aacactcgaa
     6601 gaaagggaca ggatttttaa tgcagacctt tctgccaccc cacaactggc aatacagagg
     6661 gatatattca tatttcagac actgatagga tgcagggtga gcgacctgta ccgaatgacc
     6721 aaactaaatg tggtcaatga agccatagaa tatattccca agaaaaccaa agaggggaat
     6781 ccggttacgg tacgtgttcc acttaacgac aaagcgaaag aaatccttga acgctacaaa
     6841 gaatatgagg gaaaactgtt gccgttcata tccgagcaaa agtacaatga tgccataaaa
     6901 aagatattca aattagctgg agttgaccgc atcgtaacaa tcttagaccc gttgacgcac
     6961 aacgaaatca aacgacctat ttatgaagtg gcaagcagcc atctggcaag acgtacgttt
     7021 atcggcaata tctataaaaa agtgaaagac ccgaaccttg tttccgcact gtcgggacac
     7081 aaggagggaa gcaaagcttt cagacgatac agggatattg acgaagaaat gaagaaagac
     7141 cttgtaaaac tattagactg ataaaagtat caaacagttt catagatagt t
//
