LOCUS       Exported                6101 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_50, complete sequence.
ACCESSION   MH029767
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6101)
  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 6101)
  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 6101)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6101
                     /organism="Cloning vector pTHSSe_50"
                     /mol_type="other DNA"
                     /label=incW BbsI cloning vector
                     /note="incW BbsI cloning vector"
                     /db_xref="taxon:2137931"
     regulatory      13..60
                     /regulatory_class="promoter"
                     /note="PJ23105; derived from BBa_J23105"
     ncRNA           61..135
                     /product="RiboJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     misc_RNA        63..112
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      136..154
                     /regulatory_class="ribosome_binding_site"
                     /note="B0032; derived from BBa_B0032"
     CDS             155..871
                     /codon_start=1
                     /product="sfGFP"
                     /label=sfGFP
                     /protein_id="AVR55147.1"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             155..868
                     /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      880..1008
                     /regulatory_class="terminator"
                     /note="B0015; BBa_B0015"
     terminator      888..959
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      975..1002
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1021..1041
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1042..1099
                     /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      1195..4883
                     /label=incW / pSa origin
                     /note="incW / pSa origin"
     CDS             1295..1951
                     /codon_start=1
                     /product="ResP"
                     /label=ResP
                     /note="pSa resolution protein"
                     /protein_id="AVR55145.1"
                     /translation="MPKYYAYLRVSRDGQDPENQKYGLLEYANAKGFAPLQIEEEIASR
                     AKDWRKRKLGAIIEKAERGDVLLTPEITRIAGSALAALEILKAASERGLIVHVTKQKII
                     MDGSLQSDIMATVLGLAAQIERHFIQARTTEALQVARERGKTLGRPKGSKSSALKLDSR
                     IDEVQAYVNLGLPQSRAAELLGVSPHTLRLFIKRRNIKPTNTRPTITMPGREQHA"
     CDS             1944..2915
                     /codon_start=1
                     /product="RepA"
                     /label=RepA
                     /note="pSa replication protein"
                     /protein_id="AVR55146.1"
                     /translation="MPKNNKAPGHRINEIIKTSLALEMEDAREAGLVGYMARCLVQATM
                     PHTDPKTSYFERTNGIVTLSIMGKPSIGLPYGSMPRTLLAWICTEAVRTKDPVLNLGRS
                     QSEFLQRLGMHTDGRYTATLRNQAQRLFSSMISLAGEQGNDFGIENVVIAKRAFLFWNP
                     KRPEDRALWDSTLTLTGDFFEEVTRSPVPIRIDYLHALRQSPLAMDIYTWLTYRVFLLR
                     AKGRPFVQIPWVALQAQFGSSYGSRARNSPELDDKARERAERAALASFKYNFKKRLREV
                     LIVYPEASDCIEDDGECLRIKSTRLHVTRAPGKGARIGPPPT"
     rep_origin      2952..3387
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     CDS             3633..4262
                     /codon_start=1
                     /product="KfrA"
                     /label=KfrA
                     /note="pSa partitioning protein"
                     /protein_id="AVR55148.1"
                     /translation="MAITKQDIWRAADELDAEGIRPTLAAVRKKLGSGSFTTISDAMAE
                     WKNRKTATLPSSDPLPVAVNEHLAELGNALWAIALAHANARFDEDRKQIEADKAAISQQ
                     LAEAIELADTFTRENDQLRERVNQLEPMERERDKLADQLAEVKRRSGEELNRCMEKLTQ
                     RDNEAIEARKQAKEAIERAASLQGQVEALKEQVANLTAVLKTGGKQ"
     CDS             complement(4979..5839)
                     /codon_start=1
                     /product="TEM beta-lactamase"
                     /label=TEM beta-lactamase
                     /note="ampicillin resistance protein"
                     /protein_id="AVR55144.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5840..5944)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      6034..6077
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    6080..6101
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""