LOCUS       40924_43328        6090 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_67, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6090)
  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 6090)
  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 6090)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6090)
  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..6090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      13..73
                     /label=L3S2P21
                     /note="L3S2P21"
                     /regulatory_class="terminator"
     regulatory      78..146
                     /label=PT7A1w3
                     /note="PT7A1w3"
                     /regulatory_class="promoter"
     ncRNA           147..225
                     /product="SarJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     regulatory      226..245
                     /label=RBSsp2
                     /note="RBSsp2"
                     /regulatory_class="ribosome_binding_site"
     CDS             246..2885
                     /codon_start=1
                     /product="TALEsp2"
                     /label=TALEsp2
                     /protein_id="AVR55190.1"
                     /translation="MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL
                     SQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQL
                     DTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLL
                     PVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQ
                     ALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVA
                     IASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH
                     GLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQR
                     LLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGG
                     KQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQV
                     VAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQ
                     DHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETV
                     QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNG
                     GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNR
                     RIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTEL
                     EARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMH
                     EGDQTRAS"
     regulatory      complement(2891..2980)
                     /label=ECK120029600
                     /note="ECK120029600"
                     /regulatory_class="terminator"
     regulatory      2985..3059
                     /label=PUPsp2
                     /note="PUPsp2"
                     /regulatory_class="promoter"
     misc_feature    3010..3016
                     /label=bfpT7A1
                     /note="bfpT7A1"
     protein_bind    complement(3018..3035)
                     /label=TALEsp2 binding site
                     /bound_moiety="TALEsp2"
     misc_RNA        3061..3111
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3135..3153
                     /label=RBSB03
                     /note="RBSB03"
                     /regulatory_class="ribosome_binding_site"
     misc_feature    3135..3137
                     /label=E0240
                     /note="E0240"
     CDS             3154..3867
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Nager et al., 2011)"
     terminator      3887..3958
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3974..4001
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    4020..4040
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      4041..4098
                     /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."
     rep_origin      4270..4858
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(5065..5159)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5183..5839)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(5840..5943)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     terminator      complement(6023..6066)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    6069..6090
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with
                     'ATG'"
ORIGIN
        1 gaagacacgg agctcggtac caaattccag aaaagaggcc tcccgaaagg ggggcctttt
       61 ttcgttttgg tcctactggc gcgcctcagt cagagtattg acttaaagtc taacctatag
      121 gagatctaca gccatcgaga gctgcgagac tgtcgccgga tgtgtatccg acctgacgat
      181 ggcccaaaag ggccgaaaca gtcctctaca aataattttg tttaaatcaa ttcatcgacg
      241 tgaaaatggt agatttaaga actttaggat attcacagca gcaacaggaa aagatcaagc
      301 ccaaagttag gtcgacagtc gcgcagcatc acgaagcgct ggttggtcat gggtttacac
      361 atgcccacat cgtagcctta tcgcagcacc ctgcagccct tggcacggtc gccgtcaagt
      421 accaggacat gattgcggcg ttgccggaag ccacacatga ggcgatcgtc ggtgtgggga
      481 aacagtggag cggagcccga gcgcttgagg ccctgttgac ggtcgcggga gagctgagag
      541 ggcctcccct tcagctggac acgggccagt tgctgaagat cgcgaagcgg ggaggagtca
      601 cggcggtcga ggcggtgcac gcgtggcgca atgcgctcac gggagcaccc ctcaacctga
      661 ccccggacca ggtagtcgcg attgcttcac atgacggggg taaacaagcg ctggaaacgg
      721 tgcagcgtct gctaccggtg ttatgtcagg atcatgggct cacgccggaa caggtagtgg
      781 caattgcgag tcatgacggt ggcaaacagg ccctggaaac cgtacagcgg ctgctaccgg
      841 tgctgtgtca agcgcatggc ctgactccgg accaagttgt agccattgcc tcgaacgggg
      901 gcggcaagca ggcgctggag actgttcaac gtctgctccc cgttctgtgt caggcgcatg
      961 gcctgacgcc tgcgcaggtc gtggcgatcg cttcaaacat cggtgggaag caagccctgg
     1021 agactgtcca aagactgttg ccagtgttgt gtcaagatca tggcttaacc ccagatcagg
     1081 tggttgcgat tgcatcaaat ggaggtggta aacaggcgct ggagactgtg cagcgcctgt
     1141 tgccggttct gtgccaagat catgggctga ctccggaaca ggttgtggct atcgcaagca
     1201 atattggtgg caagcaggcc ctggaaacag tacagcgcct gctgcctgta ttgtgtcaag
     1261 cccacggtct tacccccgat caggtagtcg ccatagcatc gcacgacggc gggaagcagg
     1321 cccttgagac tgtacaacgc ctcctgccgg ttttgtgcca ggcgcacggc ctgacgccag
     1381 cccaggtggt tgcgatagcc agtaataacg gcggtaagca agcccttgaa accgttcaac
     1441 gtttgctgcc agtgctgtgc caggatcacg gcctgacccc ggatcaggta gtcgccattg
     1501 ctagcaacat tggtggcaaa caagcactgg agacagttca acgcttactg cccgtgcttt
     1561 gccaggatca tggactgacc ccagagcaag tggtcgcgat tgcctcgcat gacggaggta
     1621 aacaggccct tgagactgtc cagcgtctgc tgccggtcct ttgccaggct catgggctga
     1681 cgcccgacca ggtggtagca atcgcttcga acggcggagg caaacaggcg ttagaaaccg
     1741 ttcaacgtct gttaccggtg ctgtgccaag ctcatggctt aaccccggcc caggtagtcg
     1801 caatcgctag tcatgatggt gggaaacagg cattagaaac agtacaacgt ctgctcccgg
     1861 tcctctgtca ggatcacggt ctgaccccgg atcaggttgt agcaatcgct agcaatattg
     1921 gtggtaaaca ggcgctggaa acagtgcaaa gattattacc agttctgtgt caggaccatg
     1981 gcctgacacc tgagcaggtc gtagcgatcg caagtaatat tggaggtaaa caggccctgg
     2041 aaaccgtgca gcgtctgctg cctgtgctct gtcaagcgca tggtctgact ccggatcagg
     2101 ttgtcgcgat cgccagtaac gggggaggga aacaggcact cgagactgtg cagagattgc
     2161 tgccggtcct gtgtcaggcg catggtctga ccccagcgca ggtcgtggca atcgcttcca
     2221 acataggtgg taaacaggcc ctcgaaactg tccaacggct gttaccggta ctgtgccagg
     2281 atcatggtct gacccctgag caggtagtgg ctattgcatc caacggaggg ggcagacccg
     2341 cactggagtc aatcgtggcc cagctttcga ggccggaccc cgcgctggcc gcactcacta
     2401 atgatcatct tgtagcgctg gcctgcctcg gcggacgacc cgccttggat gcggtgaaga
     2461 aggggctccc gcacgcgcct gcattgatta agcggaccaa cagaaggatt cccgagagga
     2521 catcacatcg agtggcagat cacgcgcaag tggtccgcgt gctcggattc ttccagtgtc
     2581 actcccaccc cgcacaagcg ttcgatgacg ccatgactca atttggtatg tcgagacacg
     2641 gactgctgca gctctttcgt agagtcggtg tcacagaact cgaggcccgc tcgggcacac
     2701 tgcctcccgc ctcccagcgg tgggacagga ttctccaagc gagcggtatg aaacgcgcga
     2761 agccttcacc tacgtcaact cagacacctg accaggcgag ccttcatgcg ttcgcagact
     2821 cgctggagag ggatttggac gcgccctcgc ccatgcatga aggggaccaa actcgcgcgt
     2881 cataataggt ttcagccaaa aaacttaaga ccgccggtct tgtccactac cttgcagtaa
     2941 tgcggtggac aggatcggcg gttttctttt ctcttctcaa gcttatcccg aaaatttatc
     3001 aaaaagagta ttgacttata ttgagtcgta taggatactt acagccatcg agagctgcga
     3061 gctgtcaccg gatgtgcttt ccggtctgat gagtccgtga ggacgaaaca gcctctacaa
     3121 ataattttgt ttaatcagcg cggaaagtac tagatgagca aaggagaaga acttttcact
     3181 ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc
     3241 cgtggagagg gtgaaggtga tgctacaaac ggaaaactca cccttaaatt tatttgcact
     3301 actggaaaac tacctgttcc gtggccaaca cttgtcacta ctctgaccta tggtgttcaa
     3361 tgcttttccc gttatccgga tcacatgaaa cggcatgact ttttcaagag tgccatgccc
     3421 gaaggttatg tacaggaacg cactatatct ttcaaagatg acgggaccta caagacgcgt
     3481 gctgaagtca agtttgaagg tgataccctt gttaatcgta tcgagttaaa gggtattgat
     3541 tttaaagaag atggaaacat tcttggacac aaactcgagt acaactttaa ctcacacaat
     3601 gtatacatca cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa aattcgccac
     3661 aacgttgaag atggttccgt tcaactagca gaccattatc aacaaaatac tccaattggc
     3721 gatggccctg tccttttacc agacaaccat tacctgtcga cacaatctgt cctttcgaaa
     3781 gatcccaacg aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc tgctgggatt
     3841 acacatggca tggatgagct ctacaaataa tactagagcc aggcatcaaa taaaacgaaa
     3901 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta
     3961 ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttatacgc tatgtcttct
     4021 actagtagcg gccgctgcag tccggcaaaa aagggcaagg tgtcaccacc ctgccctttt
     4081 tctttaaaac cgaaaagatt acttcgcgtt atgcaggctt cctcgctcac tgactcgctg
     4141 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aattcatgac
     4201 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4261 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4321 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4381 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4441 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4501 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4561 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4621 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4681 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4741 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4801 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4861 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4921 ctttcctgcg ttatcccctg attctgtgga taaccgtcga ttatcaaaaa ggatcttcac
     4981 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5041 ttggtctgac agctcgaggc ttggattctc accaataaaa aacgcccggc ggcaaccgag
     5101 cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca acaggagtcc
     5161 aagcgagctc gatatcaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt
     5221 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca
     5281 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga
     5341 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg
     5401 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt
     5461 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac
     5521 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac
     5581 tatcccatat caccagctca ccgtctttca ttgccatacg aaattccgga tgagcattca
     5641 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg
     5701 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg
     5761 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc
     5821 cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa
     5881 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgcccga
     5941 tcaactcgag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     6001 aataggcgta tcacgaggca gaatttcaga taaaaaaaat ccttagcttt cgctaaggat
     6061 gatttctgga attcgcggcc gcttctagag
//
