LOCUS       62056_17540        7205 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT493, complete sequence.
ACCESSION   MN991289
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7205)
  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 7205)
  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 7205)
  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..7205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41706.1"
                     /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-11"
                     /ncRNA_class="sgRNA"
     misc_RNA        3270..3345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3353..3413
                     /label=L3S3P31
                     /note="L3S3P31"
                     /regulatory_class="terminator"
     regulatory      3425..3500
                     /label=PBF2884
                     /note="PBF2884"
                     /regulatory_class="promoter"
     regulatory      3497..3500
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     misc_feature    3506..3507
                     /label=PAM
                     /note="PAM"
     ncRNA           3509..3528
                     /label=sgRNA
                     /note="sgRNA-11"
                     /ncRNA_class="sgRNA"
     misc_RNA        3534..3584
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3608..3650
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3651..4163
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      4174..4244
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      4252..4534
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      4495..4498
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4521..4528
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     CDS             4561..5172
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41709.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      5189..5249
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     primer_bind     complement(5375..5395)
                     /label=pNBU1_seq.REV
                     /note="pNBU1_seq.REV"
     misc_recomb     5493..5822
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    5620..5633
                     /label=CC Region
                     /note="CC Region"
     misc_difference 5632
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             5838..7175
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41710.1"
                     /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 aaagataaaa tggtttagtg ttttagagct agaaatagca agttaaaata
     3301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttggatcccc
     3361 aattattgaa caccctaacg ggtgtttttt tttttttggt ctcccgcgac cgcgccgcgg
     3421 ccgctgctgt cataggccgg ttgtccggta gtccggatga tttaaggaag atttctcttc
     3481 ttttttcact ttctccgaac aacctccaac taaaccattt tatctttgca gtagctgtca
     3541 ccggatgtgc tttccggtct gatgagtccg tgaggacgaa acagcctcta caaataattt
     3601 tgtttaacgc attttaaaat aaaataaatt atttatttaa ttaaacgaat atggttttta
     3661 ctctggaaga ttttgttggc gattggcgtc agaccgcggg ttataatttg gatcaagtcc
     3721 tggaacaggg tggcgtaagc tctctgttcc agaacctggg tgtgagcgtg acgccgattc
     3781 agcgcatcgt tctgtccggc gagaacggtc tgaaaattga tattcatgtg atcatcccgt
     3841 acgaaggcct gagcggtgac caaatgggtc aaatcgagaa aatctttaaa gtcgtctacc
     3901 cagttgacga tcaccacttc aaggttatct tgcattacgg tacgctggtg attgatggtg
     3961 tgaccccgaa tatgattgac tatttcggcc gtccgtatga aggcattgcc gtttttgacg
     4021 gtaaaaagat caccgtcacc ggtaccctgt ggaatggcaa taagattatt gacgagcgtc
     4081 tgattaaccc ggacggcagc ctgctgttcc gcgtgaccat caacggtgtc acgggttggc
     4141 gtctgtgcga gcgcatcctg gcataatgaa ctgggatccc tcggtaccaa attttcgaaa
     4201 aaagacgctg aaaagcgtct tttttcgttt tggtccgcgg ccgccggccg ctgatctgga
     4261 agaagcaatg aaagctgctg ttaagtctcc gaatcaggta ttgttcctga caggtgtatt
     4321 cccatccggt aaacgcggat actttgcagt tgatctgact caggaataaa ttataaatta
     4381 aggtaagaag attgtaggat aagctaatga aatagaaaaa ggatgccgtc acacaacttg
     4441 tcggcattct tttttgtttt attagttgaa aatatagtga aaaagttgcc taaatatgta
     4501 tgttaacaaa ttatttgtcg taactttgca ctccaaatct gtttttaaca tatggcacta
     4561 atgcagaaaa aattgactcg ttctcagcag aaacacttgg acatcatcaa cgctgctaaa
     4621 gaagaattca tcgaattcgg tttcttggct gctaacatgg accgtatcac ttcttctgct
     4681 gaagtatcta aacgtacttt gtaccgtcac ttcgaatcta aagaagtatt gttcgaatct
     4741 gtattgacta tcatcaacga ctctgtaaac gaatctatct cttaccactt cgacccgaac
     4801 aaatctactg aagaacagtt gactgaaatc gcttacaaag aaatcgacgt attgtacaaa
     4861 acttacggta tcgctttggc tcgtactatc gtaatggaat tcttgcgtca gccggaaatg
     4921 gctaaaactt tgatccagaa catctactct atccgtgcta tcactcagtg gttccgttct
     4981 gctatcgaag ctaaacgttt gaaagacgct gacccgaaat tgatgactga cgtatacgta
     5041 tctttgttcc agggtttgtt cttctggccg caggtaatgc acttggactt ggaaccgcac
     5101 ggtgaagaat tgtctcagaa aatcgaaact ttgactacta tcttcttgca gtcttacggt
     5161 gtagctgaat aacaatgaat gggctttcct cggtaccaaa ttccagaaaa gaggcctccc
     5221 gaaagggggg ccttttttcg ttttggtccc taattgccta tcttccagtg atggaacagc
     5281 atttgtgcat tggctgcaac aatcagcctt acttgtgcct gttctatttc cgaaccgacc
     5341 gcttgtatga atccatcaaa attcgttttc tctatgttgg attccttgtt gctcatattg
     5401 tgatgataat ttctacaaat atagtcattg gtaactatct atgaaactgt ttgatacttt
     5461 tactcatcgg attcttttaa tatcgctact ctctacgttc aaccaaaaga aataatgact
     5521 tactgctata ttttttgcac gtgtggggaa aatgtgggga aaattcaagc aaaagaaaaa
     5581 gctaagtatt gaactatcaa atacttagct ttctttcttg tacccagacc ccgcatttga
     5641 aataattaaa gtggggaaaa tgtgggtaaa aagaaaaatg cggaaaaacg ccacaattac
     5701 actgtatttc aatatgttat aatcctatta aattttaatc caagtttaat cgaattgcaa
     5761 aatatttagc agatgtgggg aaaatgctgg ggaaaatatt tatatttgca gcagagtaaa
     5821 attcaacctg aaagtctatg aaagtaacct ttatcattaa aaaagcagcc aaacgatatg
     5881 atacagaatc catggctaca atctatgtcc gttttagaaa cggaaggcag ttagactccg
     5941 ttgctcctac tcagttagcc atcaatccca atctatggga tgataaagac gaatgtgtaa
     6001 aaacgaaagc tgtctgcaat gaagaaatgc gtacccatat aaatgaagag atacgccagt
     6061 tgaaaaccta tatcgagaag gtatatcaac aagaaaagga agcaatagac aaagaatggc
     6121 taaaaacaac acttgataaa ttttaccatc ctgaaaaata ttttttgccg gaggaagtgg
     6181 ttatcaagcc taccattgga gaactattcg atgaatttct aaacaagcac cctttgtcgg
     6241 aagtacgaaa gaaaaatttc cgggttgtca aaagagcctt actgcgttat gaactatatg
     6301 taagggctac aaagagagga caaaagggct ttatccttga tgtggatttg gtaacacctg
     6361 acacgcttcg ggatatgtgg gatttctttc agaacgaata ccagtattat gaactttacc
     6421 cgagcattta tgaagccatt cccgaaaaga ggacaccaca gcccagaagc aaaaacacgc
     6481 tgatagactg tttttcaaga atacgcacat tcttcctgtg gtgcttcgat aacaaacgca
     6541 ccacaaacag acctttcgac aagtttccga tagaggagtg tacatatggt acaccttatt
     6601 atataacact cgaagaaagg gacaggattt ttaatgcaga cctttctgcc accccacaac
     6661 tggcaataca gagggatata ttcatatttc agacactgat aggatgcagg gtgagcgacc
     6721 tgtaccgaat gaccaaacta aatgtggtca atgaagccat agaatatatt cccaagaaaa
     6781 ccaaagaggg gaatccggtt acggtacgtg ttccacttaa cgacaaagcg aaagaaatcc
     6841 ttgaacgcta caaagaatat gagggaaaac tgttgccgtt catatccgag caaaagtaca
     6901 atgatgccat aaaaaagata ttcaaattag ctggagttga ccgcatcgta acaatcttag
     6961 acccgttgac gcacaacgaa atcaaacgac ctatttatga agtggcaagc agccatctgg
     7021 caagacgtac gtttatcggc aatatctata aaaaagtgaa agacccgaac cttgtttccg
     7081 cactgtcggg acacaaggag ggaagcaaag ctttcagacg atacagggat attgacgaag
     7141 aaatgaagaa agaccttgta aaactattag actgataaaa gtatcaaaca gtttcataga
     7201 tagtt
//
