pMT462 vector (V016439)

LOCUS       62056_17515        9657 bp DNA     circular SYN 19-MAY-2020
DEFINITION  Cloning vector pMT462, complete sequence.
ACCESSION   MN991284
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9657)
  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 9657)
  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 9657)
  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..9657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..890)
                     /codon_start=1
                     /transl_table=11
                     /product="ErmG"
                     /label=ErmG
                     /protein_id="QJT41682.1"
                     /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      3123..3130
                     /label=L3S3P21
                     /note="L3S3P21"
                     /regulatory_class="terminator"
     misc_feature    3134..3137
                     /label=Ascar
                     /note="Ascar"
     regulatory      3138..3245
                     /label=PM1
                     /note="PM1"
                     /regulatory_class="promoter"
     misc_RNA        3266..3341
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      3349..3452
                     /label=ECK120029600
                     /note="ECK120029600"
                     /regulatory_class="terminator"
     misc_difference 3360
                     /replace="c"
     misc_feature    3453..3456
                     /label=Bscar
                     /note="Bscar"
     protein_bind    3607..3625
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    3630..3732
                     /label=M4
                     /note="M4"
     regulatory      3733..3799
                     /label=ECK120033736
                     /note="ECK120033736"
                     /regulatory_class="terminator"
     misc_feature    3800..3803
                     /label=Dscar
                     /note="Dscar"
     regulatory      3804..3919
                     /label=P_BA
                     /note="P_BA"
                     /regulatory_class="promoter"
     misc_feature    3920..4022
                     /label=M2
                     /note="M2"
     regulatory      4023..4126
                     /label=ECK120029600
                     /note="ECK120029600"
                     /regulatory_class="terminator"
     misc_feature    4127..4130
                     /label=Escar
                     /note="Escar"
     regulatory      4131..4238
                     /label=PM1
                     /note="PM1"
                     /regulatory_class="promoter"
     misc_feature    4239..4341
                     /label=M4
                     /note="M4"
     regulatory      4342..4408
                     /label=ECK120033736
                     /note="ECK120033736"
                     /regulatory_class="terminator"
     misc_feature    4409..4412
                     /label=Fscar
                     /note="Fscar"
     regulatory      4413..4528
                     /label=P_BA
                     /note="P_BA"
                     /regulatory_class="promoter"
     misc_feature    4529..4631
                     /label=M1
                     /note="M1"
     regulatory      4632..4692
                     /label=L3S3P41
                     /note="L3S3P41"
                     /regulatory_class="terminator"
     misc_feature    4693..4696
                     /label=scar
                     /note="nonstandard type: scar"
     protein_bind    complement(4785..4803)
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    4870..4972
                     /label=M1
                     /note="M1"
     regulatory      4973..5033
                     /label=L3S3P41
                     /note="L3S3P41"
                     /regulatory_class="terminator"
     misc_feature    5034..5037
                     /label=Cscar
                     /note="Cscar"
     misc_feature    5038..5041
                     /label=Ascar
                     /note="Ascar"
     regulatory      5042..5149
                     /label=PM2
                     /note="PM2"
                     /regulatory_class="promoter"
     misc_RNA        5151..5201
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      5225..5267
                     /label=rpiL*
                     /note="rpiL*"
                     /regulatory_class="terminator"
     CDS             5268..5780
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      5791..5861
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     misc_feature    5869..5872
                     /label=Bscar
                     /note="Bscar"
     regulatory      5873..5980
                     /label=PM4
                     /note="PM4"
                     /regulatory_class="promoter"
     misc_feature    5981..6055
                     /label=RiboJ00
                     /note="RiboJ00"
     CDS             6099..6611
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
     regulatory      6622..6692
                     /label=L3S2P56
                     /note="L3S2P56"
                     /regulatory_class="terminator"
     misc_feature    6700..6703
                     /label=Cscar
                     /note="Cscar"
     regulatory      6704..6986
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="promoter"
     regulatory      6947..6950
                     /label=-33 sigma factor recognitions site
                     /note="-33 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      6973..6980
                     /label=-7 sigma factor recognitions site
                     /note="-7 sigma factor recognitions site"
                     /regulatory_class="other"
     regulatory      6987..7012
                     /label=BT1311
                     /note="BT1311"
                     /regulatory_class="ribosome_binding_site"
     CDS             7013..7624
                     /codon_start=1
                     /transl_table=11
                     /product="TetR/AcrR family transcriptional regulator"
                     /label=TetR/AcrR family transcriptional regulator
                     /note="VFA0359-Bt"
                     /protein_id="QJT41686.1"
                     /translation="MQKKLTRSQQKHLDIINAAKEEFIEFGFLAANMDRITSSAEVSKR
                     TLYRHFESKEVLFESVLTIINDSVNESISYHFDPNKSTEEQLTEIAYKEIDVLYKTYGI
                     ALARTIVMEFLRQPEMAKTLIQNIYSIRAITQWFRSAIEAKRLKDADPKLMTDVYVSLF
                     QGLFFWPQVMHLDLEPHGEELSQKIETLTTIFLQSYGVAE"
     regulatory      7641..7701
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     primer_bind     complement(7827..7847)
                     /label=pNBU1_seq.REV
                     /note="pNBU1_seq.REV"
     misc_recomb     7945..8274
                     /label=attN1 region
                     /note="attN1 region"
     misc_feature    8072..8085
                     /label=CC Region
                     /note="CC Region"
     misc_difference 8084
                     /label=mutated for higher integration efficiency
                     /note="mutated for higher integration efficiency"
     CDS             8290..9627
                     /codon_start=1
                     /transl_table=11
                     /product="IntN1"
                     /label=IntN1
                     /note="NBU1 integrase"
                     /protein_id="QJT41687.1"
                     /translation="MKVTFIIKKAAKRYDTESMATIYVRFRNGRQLDSVAPTQLAINPN
                     LWDDKDECVKTKAVCNEEMRTHINEEIRQLKTYIEKVYQQEKEAIDKEWLKTTLDKFYH
                     PEKYFLPEEVVIKPTIGELFDEFLNKHPLSEVRKKNFRVVKRALLRYELYVRATKRGQK
                     GFILDVDLVTPDTLRDMWDFFQNEYQYYELYPSIYEAIPEKRTPQPRSKNTLIDCFSRI
                     RTFFLWCFDNKRTTNRPFDKFPIEECTYGTPYYITLEERDRIFNADLSATPQLAIQRDI
                     FIFQTLIGCRVSDLYRMTKLNVVNEAIEYIPKKTKEGNPVTVRVPLNDKAKEILERYKE
                     YEGKLLPFISEQKYNDAIKKIFKLAGVDRIVTILDPLTHNEIKRPIYEVASSHLARRTF
                     IGNIYKKVKDPNLVSALSGHKEGSKAFRRYRDIDEEMKKDLVKLLD"