LOCUS       Exported                4864 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_31, complete sequence.
ACCESSION   MH029748
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4864)
  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 4864)
  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 4864)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4864
                     /organism="Cloning vector pTHSSe_31"
                     /mol_type="other DNA"
                     /label=PUPsp1 GFP reporter
                     /note="PUPsp1 GFP reporter"
                     /db_xref="taxon:2137912"
     regulatory      1..75
                     /regulatory_class="promoter"
                     /note="PUPsp1"
     protein_bind    33..50
                     /label=TALEsp1 binding site
                     /bound_moiety="TALEsp1"
     ncRNA           76..150
                     /product="RiboJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     misc_RNA        78..127
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      151..169
                     /regulatory_class="ribosome_binding_site"
                     /note="B0032; derived from BBa_B0032"
     CDS             170..886
                     /codon_start=1
                     /product="sfGFP"
                     /label=sfGFP
                     /protein_id="AVR55088.1"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             170..883
                     /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      895..1023
                     /regulatory_class="terminator"
                     /note="B0015; BBa_B0015"
     terminator      903..974
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      990..1017
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1024..1044
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1045..1102
                     /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      1257..3471
                     /label=pSC101 origin
                     /note="pSC101 origin"
     CDS             complement(1775..2725)
                     /codon_start=1
                     /product="RepA"
                     /label=RepA
                     /note="pSC101 replication protein"
                     /protein_id="AVR55090.1"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2773..2995
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      3487..3581
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3612..4472)
                     /codon_start=1
                     /product="TEM beta-lactamase"
                     /label=TEM beta-lactamase
                     /note="ampicillin resistance protein"
                     /protein_id="AVR55089.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      4797..4840
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    4843..4864
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""