LOCUS       62056_17510        7541 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT455, complete sequence.
ACCESSION   MN991283
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7541)
  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 7541)
  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 7541)
  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..7541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41677.1"
                     /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"
     protein_bind    complement(3222..3240)
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3284..3302
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      3303..3306
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     ncRNA           3307..3326
                     /label=sgRNA
                     /note="sgRNA-12"
                     /ncRNA_class="sgRNA"
     misc_RNA        3327..3402
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3416..3467
                     /label=ECK120033736
                     /note="ECK120033736"
                     /regulatory_class="terminator"
     regulatory      3476..3518
                     /label=cfxA
                     /note="cfxA"
                     /regulatory_class="promoter"
     regulatory      3532..3535
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      3543..3554
                     /label=PcfxA
                     /note="PcfxA"
                     /regulatory_class="promoter"
     regulatory      3555..3562
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     ncRNA           3592..3611
                     /label=sgRNA
                     /note="sgRNA-12"
                     /ncRNA_class="sgRNA"
     misc_feature    3612..3653
                     /label=chimera tracrRNA
                     /note="chimera tracrRNA"
     regulatory      3658..3694
                     /label=derived from S. pyogenes
                     /note="derived from S. pyogenes"
                     /regulatory_class="terminator"
     regulatory      3701..3752
                     /label=ECK120033736
                     /note="ECK120033736"
                     /regulatory_class="terminator"
     regulatory      3761..3844
                     /label=PBF2884
                     /note="PBF2884"
                     /regulatory_class="promoter"
     regulatory      3833..3836
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     misc_feature    3842..3843
                     /label=PAM
                     /note="PAM"
     ncRNA           3845..3864
                     /label=sgRNA
                     /note="sgRNA-12"
                     /ncRNA_class="sgRNA"
     misc_RNA        3870..3920
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3944..3986
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             3987..4499
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      4510..4580
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     regulatory      4588..4870
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      4831..4834
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4857..4864
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      4871..4896
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="ribosome_binding_site"
     CDS             4897..5508
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41680.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      5525..5585
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     primer_bind     complement(5711..5731)
                     /label=pNBU1_seq.REV
                     /note="pNBU1_seq.REV"
     misc_recomb     5829..6158
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    5956..5969
                     /label=CC Region
                     /note="CC Region"
     misc_difference 5968
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             6174..7511
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41681.1"
                     /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 gctttttgca cccgctttcc aagagaagaa agccttgtta aattgactta
     3181 gtgtaaaagc gcagtactgc ttgaccataa gaacaaaaaa atctctatca ctgataggga
     3241 taaagtttgg aagataaagc taaaagttct tatctttgca gtctccctat cagtgataga
     3301 gacgcacaaa gatacttatg ttagaagttt tagagctaga aatagcaagt taaaataagg
     3361 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg gatccaacgc
     3421 atgagaaagc ccccggaaga tcacctccgg gggctttttt attgcgcgcg gccgcttaca
     3481 aagaaaattc gacaaactgt tatttttcta tctatttaaa tgtacacccg tttgtttact
     3541 ttactttagt tatgtacctt tgtcggcaat gtacacccga aagtttactt tcaaagatac
     3601 ttatgttaga agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     3661 cttgaaaaag tggcaccgag tcggtgcttt ttttggatcc aacgcatgag aaagcccccg
     3721 gaagatcacc tccgggggct tttttattgc gcgcggccgc tgctgtcata ggccggttgt
     3781 ccggtagtcc ggatgattta aggaagattt ctcttctttt ttcactttct ccgaacaacc
     3841 tccattctaa cataagtatc tttgcagtag ctgtcaccgg atgtgctttc cggtctgatg
     3901 agtccgtgag gacgaaacag cctctacaaa taattttgtt taacgcattt taaaataaaa
     3961 taaattattt atttaattaa acgaatatgg tttttactct ggaagatttt gttggcgatt
     4021 ggcgtcagac cgcgggttat aatttggatc aagtcctgga acagggtggc gtaagctctc
     4081 tgttccagaa cctgggtgtg agcgtgacgc cgattcagcg catcgttctg tccggcgaga
     4141 acggtctgaa aattgatatt catgtgatca tcccgtacga aggcctgagc ggtgaccaaa
     4201 tgggtcaaat cgagaaaatc tttaaagtcg tctacccagt tgacgatcac cacttcaagg
     4261 ttatcttgca ttacggtacg ctggtgattg atggtgtgac cccgaatatg attgactatt
     4321 tcggccgtcc gtatgaaggc attgccgttt ttgacggtaa aaagatcacc gtcaccggta
     4381 ccctgtggaa tggcaataag attattgacg agcgtctgat taacccggac ggcagcctgc
     4441 tgttccgcgt gaccatcaac ggtgtcacgg gttggcgtct gtgcgagcgc atcctggcat
     4501 aatgaactgg gatccctcgg taccaaattt tcgaaaaaag acgctgaaaa gcgtcttttt
     4561 tcgttttggt ccgcggccgc cggccgctga tctggaagaa gcaatgaaag ctgctgttaa
     4621 gtctccgaat caggtattgt tcctgacagg tgtattccca tccggtaaac gcggatactt
     4681 tgcagttgat ctgactcagg aataaattat aaattaaggt aagaagattg taggataagc
     4741 taatgaaata gaaaaaggat gccgtcacac aacttgtcgg cattcttttt tgttttatta
     4801 gttgaaaata tagtgaaaaa gttgcctaaa tatgtatgtt aacaaattat ttgtcgtaac
     4861 tttgcactcc aaatctgttt ttaacatatg gcactaatgc agaaaaaatt gactcgttct
     4921 cagcagaaac acttggacat catcaacgct gctaaagaag aattcatcga attcggtttc
     4981 ttggctgcta acatggaccg tatcacttct tctgctgaag tatctaaacg tactttgtac
     5041 cgtcacttcg aatctaaaga agtattgttc gaatctgtat tgactatcat caacgactct
     5101 gtaaacgaat ctatctctta ccacttcgac ccgaacaaat ctactgaaga acagttgact
     5161 gaaatcgctt acaaagaaat cgacgtattg tacaaaactt acggtatcgc tttggctcgt
     5221 actatcgtaa tggaattctt gcgtcagccg gaaatggcta aaactttgat ccagaacatc
     5281 tactctatcc gtgctatcac tcagtggttc cgttctgcta tcgaagctaa acgtttgaaa
     5341 gacgctgacc cgaaattgat gactgacgta tacgtatctt tgttccaggg tttgttcttc
     5401 tggccgcagg taatgcactt ggacttggaa ccgcacggtg aagaattgtc tcagaaaatc
     5461 gaaactttga ctactatctt cttgcagtct tacggtgtag ctgaataaca atgaatgggc
     5521 tttcctcggt accaaattcc agaaaagagg cctcccgaaa ggggggcctt ttttcgtttt
     5581 ggtccctaat tgcctatctt ccagtgatgg aacagcattt gtgcattggc tgcaacaatc
     5641 agccttactt gtgcctgttc tatttccgaa ccgaccgctt gtatgaatcc atcaaaattc
     5701 gttttctcta tgttggattc cttgttgctc atattgtgat gataatttct acaaatatag
     5761 tcattggtaa ctatctatga aactgtttga tacttttact catcggattc ttttaatatc
     5821 gctactctct acgttcaacc aaaagaaata atgacttact gctatatttt ttgcacgtgt
     5881 ggggaaaatg tggggaaaat tcaagcaaaa gaaaaagcta agtattgaac tatcaaatac
     5941 ttagctttct ttcttgtacc cagaccccgc atttgaaata attaaagtgg ggaaaatgtg
     6001 ggtaaaaaga aaaatgcgga aaaacgccac aattacactg tatttcaata tgttataatc
     6061 ctattaaatt ttaatccaag tttaatcgaa ttgcaaaata tttagcagat gtggggaaaa
     6121 tgctggggaa aatatttata tttgcagcag agtaaaattc aacctgaaag tctatgaaag
     6181 taacctttat cattaaaaaa gcagccaaac gatatgatac agaatccatg gctacaatct
     6241 atgtccgttt tagaaacgga aggcagttag actccgttgc tcctactcag ttagccatca
     6301 atcccaatct atgggatgat aaagacgaat gtgtaaaaac gaaagctgtc tgcaatgaag
     6361 aaatgcgtac ccatataaat gaagagatac gccagttgaa aacctatatc gagaaggtat
     6421 atcaacaaga aaaggaagca atagacaaag aatggctaaa aacaacactt gataaatttt
     6481 accatcctga aaaatatttt ttgccggagg aagtggttat caagcctacc attggagaac
     6541 tattcgatga atttctaaac aagcaccctt tgtcggaagt acgaaagaaa aatttccggg
     6601 ttgtcaaaag agccttactg cgttatgaac tatatgtaag ggctacaaag agaggacaaa
     6661 agggctttat ccttgatgtg gatttggtaa cacctgacac gcttcgggat atgtgggatt
     6721 tctttcagaa cgaataccag tattatgaac tttacccgag catttatgaa gccattcccg
     6781 aaaagaggac accacagccc agaagcaaaa acacgctgat agactgtttt tcaagaatac
     6841 gcacattctt cctgtggtgc ttcgataaca aacgcaccac aaacagacct ttcgacaagt
     6901 ttccgataga ggagtgtaca tatggtacac cttattatat aacactcgaa gaaagggaca
     6961 ggatttttaa tgcagacctt tctgccaccc cacaactggc aatacagagg gatatattca
     7021 tatttcagac actgatagga tgcagggtga gcgacctgta ccgaatgacc aaactaaatg
     7081 tggtcaatga agccatagaa tatattccca agaaaaccaa agaggggaat ccggttacgg
     7141 tacgtgttcc acttaacgac aaagcgaaag aaatccttga acgctacaaa gaatatgagg
     7201 gaaaactgtt gccgttcata tccgagcaaa agtacaatga tgccataaaa aagatattca
     7261 aattagctgg agttgaccgc atcgtaacaa tcttagaccc gttgacgcac aacgaaatca
     7321 aacgacctat ttatgaagtg gcaagcagcc atctggcaag acgtacgttt atcggcaata
     7381 tctataaaaa agtgaaagac ccgaaccttg tttccgcact gtcgggacac aaggagggaa
     7441 gcaaagcttt cagacgatac agggatattg acgaagaaat gaagaaagac cttgtaaaac
     7501 tattagactg ataaaagtat caaacagttt catagatagt t
//
