LOCUS       Exported                7544 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_60, complete sequence.
ACCESSION   MH029777
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7544)
  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 7544)
  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 7544)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7544
                     /organism="Cloning vector pTHSSe_60"
                     /mol_type="other DNA"
                     /label=TALEsp2 stabilized promoter driving GFP
                     /note="TALEsp2 stabilized promoter driving GFP"
                     /db_xref="taxon:2137941"
     regulatory      13..73
                     /regulatory_class="terminator"
                     /note="L3S2P21"
     regulatory      78..146
                     /regulatory_class="promoter"
                     /note="PT7A1w3"
     ncRNA           147..225
                     /product="SarJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     regulatory      226..245
                     /regulatory_class="ribosome_binding_site"
                     /note="RBSsp2"
     CDS             246..2885
                     /codon_start=1
                     /product="TALEsp2"
                     /label=TALEsp2
                     /protein_id="AVR55170.1"
                     /translation="MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL
                     SQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQL
                     DTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLL
                     PVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQ
                     ALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVA
                     IASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH
                     GLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQR
                     LLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGG
                     KQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQV
                     VAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQ
                     DHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETV
                     QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNG
                     GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNR
                     RIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTEL
                     EARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMH
                     EGDQTRAS"
     regulatory      complement(2891..2980)
                     /regulatory_class="terminator"
                     /note="ECK120029600"
     regulatory      2985..3059
                     /regulatory_class="promoter"
                     /note="PUPsp2"
     misc_feature    3010..3016
                     /label=bfpT7A1
                     /note="bfpT7A1"
     protein_bind    complement(3018..3035)
                     /label=TALEsp2 binding site
                     /bound_moiety="TALEsp2"
     ncRNA           3060..3134
                     /product="RiboJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     misc_RNA        3062..3111
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3135..3153
                     /regulatory_class="ribosome_binding_site"
                     /note="B0032; derived from BBa_B0032"
     CDS             3154..3870
                     /codon_start=1
                     /product="sfGFP"
                     /label=sfGFP
                     /protein_id="AVR55168.1"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             3154..3867
                     /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      3879..4007
                     /regulatory_class="terminator"
                     /note="B0015; BBa_B0015"
     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      complement(4197..6411)
                     /direction=LEFT
                     /label=pSC101 origin
                     /note="pSC101 origin"
     rep_origin      4673..4895
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4943..5893
                     /codon_start=1
                     /product="RepA"
                     /label=RepA
                     /note="pSC101 replication protein"
                     /protein_id="AVR55169.1"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     terminator      6519..6613
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     misc_feature    complement(6634..7293)
                     /label=similar to chloramphenicol resistance protein
                     /note="similar to chloramphenicol resistance protein"
     CDS             complement(6634..7293)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     terminator      7477..7520
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    7523..7544
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""