LOCUS       40924_43273        6896 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_40, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6896)
  AUTHORS   Segall-Shapiro TH, Sontag ED, Voigt CA.
  TITLE     Engineered promoters enable constant gene expression at any copy 
            number in bacteria
  JOURNAL   Nat. Biotechnol. (2018) In press
  PUBMED    29553576
REFERENCE   2  (bases 1 to 6896)
  AUTHORS   Segall-Shapiro TH, Sontag ED, Voigt CA.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-MAR-2018) Synthetic Biology Center, Department of 
            Biological Engineering, Massachusetts Institute of Technology, 77 
            Massachusetts Avenue, Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 6896)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6896)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Biotechnol. (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (06-MAR-2018) Synthetic Biology Center, Department of Biological 
            Engineering, Massachusetts Institute of Technology, 77 Massachusetts
            Avenue, Cambridge, MA 02139, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6896
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..31
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    complement(37..56)
                     /label=lac operator (symmetric)
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     misc_RNA        73..123
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      147..165
                     /note="RBS32A; derived from BBa_B0032"
                     /regulatory_class="ribosome_binding_site"
     CDS             166..2805
                     /codon_start=1
                     /product="TALEsp2"
                     /label=TALEsp2
                     /protein_id="AVR55114.1"
                     /translation="MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL
                     SQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQL
                     DTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLL
                     PVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQ
                     ALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVA
                     IASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH
                     GLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQR
                     LLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGG
                     KQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQV
                     VAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQ
                     DHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETV
                     QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNG
                     GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNR
                     RIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTEL
                     EARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMH
                     EGDQTRAS"
     terminator      2819..2890
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      2906..2933
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    2940..2960
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      2961..3018
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E.
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     CDS             complement(3179..3967)
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     rep_origin      complement(4562..5106)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     terminator      complement(5596..5639)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(5685..6764)
                     /label=lacI
                     /note="lac repressor"
     regulatory      complement(6765..6846)
                     /label=LacI promoter
                     /note="LacI promoter"
                     /regulatory_class="promoter"
ORIGIN
        1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattctat
       61 ggactatgtt tagctgtcac cggatgtgct ttccggtctg atgagtccgt gaggacgaaa
      121 cagcctctac aaataatttt gtttaatcac acaggaaagt actaaatggt agatttaaga
      181 actttaggat attcacagca gcaacaggaa aagatcaagc ccaaagttag gtcgacagtc
      241 gcgcagcatc acgaagcgct ggttggtcat gggtttacac atgcccacat cgtagcctta
      301 tcgcagcacc ctgcagccct tggcacggtc gccgtcaagt accaggacat gattgcggcg
      361 ttgccggaag ccacacatga ggcgatcgtc ggtgtgggga aacagtggag cggagcccga
      421 gcgcttgagg ccctgttgac ggtcgcggga gagctgagag ggcctcccct tcagctggac
      481 acgggccagt tgctgaagat cgcgaagcgg ggaggagtca cggcggtcga ggcggtgcac
      541 gcgtggcgca atgcgctcac gggagcaccc ctcaacctga ccccggacca ggtagtcgcg
      601 attgcttcac atgacggggg taaacaagcg ctggaaacgg tgcagcgtct gctaccggtg
      661 ttatgtcagg atcatgggct cacgccggaa caggtagtgg caattgcgag tcatgacggt
      721 ggcaaacagg ccctggaaac cgtacagcgg ctgctaccgg tgctgtgtca agcgcatggc
      781 ctgactccgg accaagttgt agccattgcc tcgaacgggg gcggcaagca ggcgctggag
      841 actgttcaac gtctgctccc cgttctgtgt caggcgcatg gcctgacgcc tgcgcaggtc
      901 gtggcgatcg cttcaaacat cggtgggaag caagccctgg agactgtcca aagactgttg
      961 ccagtgttgt gtcaagatca tggcttaacc ccagatcagg tggttgcgat tgcatcaaat
     1021 ggaggtggta aacaggcgct ggagactgtg cagcgcctgt tgccggttct gtgccaagat
     1081 catgggctga ctccggaaca ggttgtggct atcgcaagca atattggtgg caagcaggcc
     1141 ctggaaacag tacagcgcct gctgcctgta ttgtgtcaag cccacggtct tacccccgat
     1201 caggtagtcg ccatagcatc gcacgacggc gggaagcagg cccttgagac tgtacaacgc
     1261 ctcctgccgg ttttgtgcca ggcgcacggc ctgacgccag cccaggtggt tgcgatagcc
     1321 agtaataacg gcggtaagca agcccttgaa accgttcaac gtttgctgcc agtgctgtgc
     1381 caggatcacg gcctgacccc ggatcaggta gtcgccattg ctagcaacat tggtggcaaa
     1441 caagcactgg agacagttca acgcttactg cccgtgcttt gccaggatca tggactgacc
     1501 ccagagcaag tggtcgcgat tgcctcgcat gacggaggta aacaggccct tgagactgtc
     1561 cagcgtctgc tgccggtcct ttgccaggct catgggctga cgcccgacca ggtggtagca
     1621 atcgcttcga acggcggagg caaacaggcg ttagaaaccg ttcaacgtct gttaccggtg
     1681 ctgtgccaag ctcatggctt aaccccggcc caggtagtcg caatcgctag tcatgatggt
     1741 gggaaacagg cattagaaac agtacaacgt ctgctcccgg tcctctgtca ggatcacggt
     1801 ctgaccccgg atcaggttgt agcaatcgct agcaatattg gtggtaaaca ggcgctggaa
     1861 acagtgcaaa gattattacc agttctgtgt caggaccatg gcctgacacc tgagcaggtc
     1921 gtagcgatcg caagtaatat tggaggtaaa caggccctgg aaaccgtgca gcgtctgctg
     1981 cctgtgctct gtcaagcgca tggtctgact ccggatcagg ttgtcgcgat cgccagtaac
     2041 gggggaggga aacaggcact cgagactgtg cagagattgc tgccggtcct gtgtcaggcg
     2101 catggtctga ccccagcgca ggtcgtggca atcgcttcca acataggtgg taaacaggcc
     2161 ctcgaaactg tccaacggct gttaccggta ctgtgccagg atcatggtct gacccctgag
     2221 caggtagtgg ctattgcatc caacggaggg ggcagacccg cactggagtc aatcgtggcc
     2281 cagctttcga ggccggaccc cgcgctggcc gcactcacta atgatcatct tgtagcgctg
     2341 gcctgcctcg gcggacgacc cgccttggat gcggtgaaga aggggctccc gcacgcgcct
     2401 gcattgatta agcggaccaa cagaaggatt cccgagagga catcacatcg agtggcagat
     2461 cacgcgcaag tggtccgcgt gctcggattc ttccagtgtc actcccaccc cgcacaagcg
     2521 ttcgatgacg ccatgactca atttggtatg tcgagacacg gactgctgca gctctttcgt
     2581 agagtcggtg tcacagaact cgaggcccgc tcgggcacac tgcctcccgc ctcccagcgg
     2641 tgggacagga ttctccaagc gagcggtatg aaacgcgcga agccttcacc tacgtcaact
     2701 cagacacctg accaggcgag ccttcatgcg ttcgcagact cgctggagag ggatttggac
     2761 gcgccctcgc ccatgcatga aggggaccaa actcgcgcgt cataataggt ccaggcatca
     2821 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     2881 gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct gcgtttatat
     2941 actagtagcg gccgctgcag tccggcaaaa aagggcaagg tgtcaccacc ctgccctttt
     3001 tctttaaaac cgaaaagatt acttcgcgtt atgcaggctt cctcgctcac tgactcgctg
     3061 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatctcgagt
     3121 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattatt
     3181 tgccgactac cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg
     3241 tgcgcgaggc caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga
     3301 cgggctgata ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg
     3361 cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct
     3421 catcgccagc ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa
     3481 atagatcctg ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa
     3541 cgctatgttc tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct
     3601 cgaagatacc tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag
     3661 ctggataacg ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga
     3721 gaatctcgct ctctacaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc
     3781 gcgttgtttc atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct
     3841 tcaggccgcc atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat
     3901 ggcgctcgat gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt
     3961 ccctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg
     4021 atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg tggctttgtt
     4081 gaataaatcg aacttttgct gagttgaagg atcagatcac gcatcttccc gacaacgcag
     4141 accgttccgt ggcaaagcaa aagttcaaaa tcaccaactg gtccacctac aacaaagctc
     4201 tcatcaaccg tggctccctc actttctggc tggatgatgg ggcgattcag gcctggtatg
     4261 agtcagcaac accttcttca cgaggcagac ctcagcgcta gcggagtgta tactggctta
     4321 ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct
     4381 gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc gctcactgac
     4441 tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat
     4501 ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt
     4561 ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt
     4621 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc cctggcggct ccctcgtgcg
     4681 ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc
     4741 tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc
     4801 acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4861 acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag
     4921 ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc
     4981 gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa
     5041 ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga
     5101 tctcaagaag atcatcttat taaggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5161 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5221 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5281 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5341 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5401 atgataccgc gatacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5461 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattcca
     5521 tggtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5581 gtatcacgag gcagaatttc agataaaaaa aatccttagc tttcgctaag gatgatttct
     5641 ggtcggccaa ttcgcgctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5701 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5761 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agactggcaa cagctgattg
     5821 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     5881 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct atcttcggta
     5941 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     6001 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     6061 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     6121 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6181 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6241 accagatgct ccacgcccag tcgcgtaccg tcctcatggg agaaaataat actgttgatg
     6301 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6361 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6421 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6481 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6541 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6601 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6661 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6721 acaccggcat actctgcgac atcgtataac gttactggtt tcatattcac caccctgaat
     6781 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggcg
     6841 cgccgcttcg tcaggccaca tagctttctt gttctgatcg gaacgatcgt tggctg
//
