LOCUS       Exported                7538 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_61, complete sequence.
ACCESSION   MH029778
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7538)
  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 7538)
  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 7538)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7538
                     /organism="Cloning vector pTHSSe_61"
                     /mol_type="other DNA"
                     /note="TALEsp1 weak1(Psp1) stabilized promoter driving GFP"
                     /db_xref="taxon:2137942"
     regulatory      13..73
                     /regulatory_class="terminator"
                     /note="L3S2P21"
     regulatory      78..146
                     /regulatory_class="promoter"
                     /note="PT7A1w2"
     ncRNA           147..225
                     /product="SarJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     regulatory      226..245
                     /regulatory_class="ribosome_binding_site"
                     /note="RBSsp1"
     CDS             246..2885
                     /codon_start=1
                     /product="TALEsp1"
                     /label=TALEsp1
                     /protein_id="AVR55173.1"
                     /translation="MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL
                     SQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQL
                     DTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLL
                     PVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQ
                     ALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVA
                     IASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAH
                     GLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQR
                     LLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGG
                     KQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQV
                     VAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQ
                     DHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETV
                     QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNN
                     GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNR
                     RIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTEL
                     EARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMH
                     EGDQTRAS"
     regulatory      complement(2891..2980)
                     /regulatory_class="terminator"
                     /note="ECK120029600"
     regulatory      2985..3053
                     /regulatory_class="promoter"
                     /note="Psp1"
     protein_bind    3011..3028
                     /label=TALEsp1 binding site
                     /bound_moiety="TALEsp1"
     ncRNA           3054..3128
                     /product="RiboJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     misc_RNA        3056..3105
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3129..3147
                     /regulatory_class="ribosome_binding_site"
                     /note="B0032; derived from BBa_B0032"
     CDS             3148..3864
                     /codon_start=1
                     /product="sfGFP"
                     /label=sfGFP
                     /protein_id="AVR55171.1"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             3148..3861
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     regulatory      3873..4001
                     /regulatory_class="terminator"
                     /note="B0015; BBa_B0015"
     terminator      3881..3952
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3968..3995
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    4014..4034
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      4035..4092
                     /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      complement(4191..6405)
                     /direction=LEFT
                     /label=pSC101 origin
                     /note="pSC101 origin"
     rep_origin      4667..4889
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4937..5887
                     /codon_start=1
                     /product="RepA"
                     /label=RepA
                     /note="pSC101 replication protein"
                     /protein_id="AVR55172.1"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     terminator      6513..6607
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     misc_feature    complement(6628..7287)
                     /label=similar to chloramphenicol resistance protein
                     /note="similar to chloramphenicol resistance protein"
     CDS             complement(6628..7287)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     terminator      7471..7514
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    7517..7538
                     /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 gaccgttaca gccatcgaga gctgcgagac tgtcgccgga tgtgtatccg acctgacgat
      181 ggcccaaaag ggccgaaaca gtcctctaca aataattttg tttaaatcaa ttcacctgcg
      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 ctcaatctga
      661 ccccggatca ggtggttgcg atcgccagta atggcggagg caaacaagca ctcgaaactg
      721 tacagcgcct cctgccggta ctgtgccaag atcatggctt gacgcctgag caagtcgtag
      781 ctattgcatc aaacatcggt ggtaaacagg cgctggaaac cgtacaacga ttactccctg
      841 tcttatgcca ggcacacggt ctgacccccg accaagttgt agccattgcg tctaatggcg
      901 gcgggaaaca ggccctggaa acggtccaac gtctgttacc cgttctgtgt caggctcacg
      961 gtctgacccc tgcccaggta gttgcaattg ccagcaacat cggcgggaaa caagcgctgg
     1021 agactgtgca gcgtctgctc cctgtgttat gccaagatca tgggctcact ccggatcagg
     1081 tggtggccat cgcttccaat attggcggta aacaggcgct ggagacagtg caacgacttt
     1141 tacctgttct ctgccaggat catggtctaa ctcccgagca ggtcgtcgcc atcgcctctc
     1201 atgacggcgg gaaacaagcg ttggaaactg tccagcgact cctgccggtt ttgtgccagg
     1261 cccacgggct tactcctgac caggtagttg cgatcgcgtc aaatgggggt ggcaaacaag
     1321 ccctcgaaac cgtgcaacgc ctgctgcccg tcttgtgcca agctcatggg ctgactccgg
     1381 cgcaagtagt cgcgattgcg agccacgatg gcggtaagca ggcactggaa acggttcagc
     1441 gcctgctccc ggttctatgc caggatcacg gcctgacccc ggaccaggtc gtcgcgatcg
     1501 cgtcaaatat cggtggcaaa caagctttgg agacagtaca gcgcctgtta ccagtgcttt
     1561 gccaggacca tggtctgacc cctgagcaag tagtggcgat cgcttctaat attgggggca
     1621 aacaagcgct ggaaacagta cagcgtctgt taccggtcct atgccaggca catggcctga
     1681 cccctgatca ggtggtagcc attgccagtc atgatggcgg taaacaggcg cttgagactg
     1741 tccaacgtct gctgccggtc ctctgtcagg ctcatggcct gacgccagct caagtcgtgg
     1801 ctatcgcttc gcatgatggc ggaaaacagg cactggagac tgtgcagcga ctgttgccag
     1861 ttctgtgtca ggatcacggt ttaactccgg accaggtggt cgctattgcg tcgaatgggg
     1921 gcggtaaaca agcgctggaa actgtgcaac gtttgctccc agttctgtgc caggaccatg
     1981 ggctgactcc ggaacaggta gtggccattg cttctaatat tggtgggaaa caggcgctgg
     2041 aaaccgtgca gcgcctgctt ccagtgcttt gccaggccca tggcctgacg ccagatcagg
     2101 tggttgctat agccagcaat ggcggcggta aacaggccct cgaaaccgtc cagcgcctgc
     2161 tccctgtgct gtgccaggcc catgggctta ccccagcgca agtagtggcg attgcgtcta
     2221 atattggtgg taaacaggcg ttggagactg tacaacgcct gctgccagtt ttatgccaag
     2281 atcatggtct gacccctgag caggtagtgg ctattgcatc caacaacggg 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 gcttggcgcg cctcaaaaag
     3001 agtattgact tataactcaa cctataggat acttacagcc atcgagagct gcgagctgtc
     3061 accggatgtg ctttccggtc tgatgagtcc gtgaggacga aacagcctct acaaataatt
     3121 ttgtttaatc acacaggaaa gtactagatg agcaaaggag aagaactttt cactggagtt
     3181 gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtccgtgga
     3241 gagggtgaag gtgatgctac aaacggaaaa ctcaccctta aatttatttg cactactgga
     3301 aaactacctg ttccgtggcc aacacttgtc actactctga cctatggtgt tcaatgcttt
     3361 tcccgttatc cggatcacat gaaacggcat gactttttca agagtgccat gcccgaaggt
     3421 tatgtacagg aacgcactat atctttcaaa gatgacggga cctacaagac gcgtgctgaa
     3481 gtcaagtttg aaggtgatac ccttgttaat cgtatcgagt taaagggtat tgattttaaa
     3541 gaagatggaa acattcttgg acacaaactc gagtacaact ttaactcaca caatgtatac
     3601 atcacggcag acaaacaaaa gaatggaatc aaagctaact tcaaaattcg ccacaacgtt
     3661 gaagatggtt ccgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggc
     3721 cctgtccttt taccagacaa ccattacctg tcgacacaat ctgtcctttc gaaagatccc
     3781 aacgaaaagc gtgaccacat ggtccttctt gagtttgtaa ctgctgctgg gattacacat
     3841 ggcatggatg agctctacaa ataatactag agccaggcat caaataaaac gaaaggctca
     3901 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag
     3961 tcacactggc tcaccttcgg gtgggccttt ctgcgtttat acgctatgtc ttctactagt
     4021 agcggccgct gcagtccggc aaaaaagggc aaggtgtcac caccctgccc tttttcttta
     4081 aaaccgaaaa gattacttcg cgttatgcag gcttcctcgc tcactgactc gctgcgctcg
     4141 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatgac agtaagacgg
     4201 gtaagcctgt tgatgatacc gctgccttac tgggtgcatt agccagtctg aatgacctgt
     4261 cacgggataa tccgaagtgg tcagactgga aaatcagagg gcaggaactg ctgaacagca
     4321 aaaagtcaga tagcaccaca tagcagaccc gccataaaac gccctgagaa gcccgtgacg
     4381 ggcttttctt gtattatggg tagtttcctt gcatgaatcc ataaaaggcg cctgtagtgc
     4441 catttacccc cattcactgc cagagccgtg agcgcagcga actgaatgtc acgaaaaaga
     4501 cagcgactca ggtgcctgat ggtcggagac aaaaggaata ttcagcgatt tgcccgagct
     4561 tgcgagggtg ctacttaagc ctttagggtt ttaaggtctg ttttgtagag gagcaaacag
     4621 cgtttgcgac atccttttgt aatactgcgg aactgactaa agtagtgagt tatacacagg
     4681 gctgggatct attcttttta tcttttttta ttctttcttt attctataaa ttataaccac
     4741 ttgaatataa acaaaaaaaa cacacaaagg tctagcggaa tttacagagg gtctagcaga
     4801 atttacaagt tttccagcaa aggtctagca gaatttacag atacccacaa ctcaaaggaa
     4861 aaggactagt aattatcatt gactagccca tctcaattgg tatagtgatt aaaatcacct
     4921 agaccaattg agatgtatgt ctgaattagt tgttttcaaa gcaaatgaac tagcgattag
     4981 tcgctatgac ttaacggagc atgaaaccaa gctaatttta tgctgtgtgg cactactcaa
     5041 ccccacgatt gaaaacccta caaggaaaga acggacggta tcgttcactt ataaccaata
     5101 cgctcagatg atgaacatca gtagggaaaa tgcttatggt gtattagcta aagcaaccag
     5161 agagctgatg acgagaactg tggaaatcag gaatcctttg gttaaaggct ttgagatttt
     5221 ccagtggaca aactatgcca agttctcaag cgaaaaatta gaattagttt ttagtgaaga
     5281 gatattgcct tatcttttcc agttaaaaaa attcataaaa tataatctgg aacatgttaa
     5341 gtcttttgaa aacaaatact ctatgaggat ttatgagtgg ttattaaaag aactaacaca
     5401 aaagaaaact cacaaggcaa atatagagat tagccttgat gaatttaagt tcatgttaat
     5461 gcttgaaaat aactaccatg agtttaaaag gcttaaccaa tgggttttga aaccaataag
     5521 taaagattta aacacttaca gcaatatgaa attggtggtt gataagcgag gccgcccgac
     5581 tgatacgttg attttccaag ttgaactaga tagacaaatg gatctcgtaa ccgaacttga
     5641 gaacaaccag ataaaaatga atggtgacaa aataccaaca accattacat cagattccta
     5701 cctacataac ggactaagaa aaacactaca cgatgcttta actgcaaaaa ttcagctcac
     5761 cagttttgag gcaaaatttt tgagtgacat gcaaagtaag tatgatctca atggttcgtt
     5821 ctcatggctc acgcaaaaac aacgaaccac actagagaac atactggcta aatacggaag
     5881 gatctgaggt tcttatggct cttgtatcta tcagtgaagc atcaagacta acaaacaaaa
     5941 gtagaacaac tgttcaccgt tacatatcaa agggaaaact gtccatatgc acagatgaaa
     6001 acggtgtaaa aaagatagat acatcagagc ttttacgagt ttttggtgca ttcaaagctg
     6061 ttcaccatga acagatcgac aatgtaacag atgaacagca tgtaacacct aatagaacag
     6121 gtgaaaccag taaaacaaag caactagaac atgaaattga acacctgaga caacttgtta
     6181 cagctcaaca gtcacacata gacagcctga aacaggcgat gctgcttatc gaatcaaagc
     6241 tgccgacaac acgggagcca gtgacgcctc ccgtggggaa aaaatcatgg caattctgga
     6301 agaaatagcg ctttcagccg gcaaaccggc tgaagccgga tctgcgattc tgataacaaa
     6361 ctagcaacac cagaacagcc cgtttgcggg cagcaaaacc cgtaccgatt atcaaaaagg
     6421 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6481 gagtaaactt ggtctgacag ctcgaggctt ggattctcac caataaaaaa cgcccggcgg
     6541 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     6601 aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg
     6661 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     6721 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     6781 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     6841 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     6901 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     6961 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     7021 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg
     7081 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     7141 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     7201 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     7261 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa
     7321 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac
     7381 gtgcccgatc aactcgagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     7441 cctataaaaa taggcgtatc acgaggcaga atttcagata aaaaaaatcc ttagctttcg
     7501 ctaaggatga tttctggaat tcgcggccgc ttctagag
//
