LOCUS       Exported                4856 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_21, complete sequence.
ACCESSION   MH029738
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4856)
  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 4856)
  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 4856)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4856
                     /organism="Cloning vector pTHSSe_21"
                     /mol_type="other DNA"
                     /label=PTALE1(-3)R GFP reporter
                     /note="PTALE1(-3)R GFP reporter"
                     /db_xref="taxon:2137902"
     regulatory      1..67
                     /regulatory_class="promoter"
                     /note="PTALE1(-3)R"
     protein_bind    complement(54..67)
                     /label=TALE1 binding site
                     /bound_moiety="TALE1"
     ncRNA           68..142
                     /product="RiboJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     misc_RNA        70..119
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      143..161
                     /regulatory_class="ribosome_binding_site"
                     /note="B0032; derived from BBa_B0032"
     CDS             162..878
                     /codon_start=1
                     /product="sfGFP"
                     /label=sfGFP
                     /protein_id="AVR55060.1"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             162..875
                     /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      887..1015
                     /regulatory_class="terminator"
                     /note="B0015; BBa_B0015"
     terminator      895..966
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      982..1009
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1016..1036
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1037..1094
                     /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      1249..3463
                     /label=pSC101 origin
                     /note="pSC101 origin"
     CDS             complement(1767..2717)
                     /codon_start=1
                     /product="RepA"
                     /label=RepA
                     /note="pSC101 replication protein"
                     /protein_id="AVR55059.1"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2765..2987
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      3479..3573
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3604..4464)
                     /codon_start=1
                     /product="TEM beta-lactamase"
                     /label=TEM beta-lactamase
                     /note="ampicillin resistance protein"
                     /protein_id="AVR55058.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      4789..4832
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    4835..4856
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
ORIGIN
        1 ggcgcgcctc aaaaagagta ttgacttaaa gtctaaccta taggatactt acatatagtg
       61 agtcgtaagc tgtcaccgga tgtgctttcc ggtctgatga gtccgtgagg acgaaacagc
      121 ctctacaaat aattttgttt aatcacacag gaaagtacta gatgagcaaa ggagaagaac
      181 ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat
      241 tttctgtccg tggagagggt gaaggtgatg ctacaaacgg aaaactcacc cttaaattta
      301 tttgcactac tggaaaacta cctgttccgt ggccaacact tgtcactact ctgacctatg
      361 gtgttcaatg cttttcccgt tatccggatc acatgaaacg gcatgacttt ttcaagagtg
      421 ccatgcccga aggttatgta caggaacgca ctatatcttt caaagatgac gggacctaca
      481 agacgcgtgc tgaagtcaag tttgaaggtg atacccttgt taatcgtatc gagttaaagg
      541 gtattgattt taaagaagat ggaaacattc ttggacacaa actcgagtac aactttaact
      601 cacacaatgt atacatcacg gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa
      661 ttcgccacaa cgttgaagat ggttccgttc aactagcaga ccattatcaa caaaatactc
      721 caattggcga tggccctgtc cttttaccag acaaccatta cctgtcgaca caatctgtcc
      781 tttcgaaaga tcccaacgaa aagcgtgacc acatggtcct tcttgagttt gtaactgctg
      841 ctgggattac acatggcatg gatgagctct acaaataata ctagagccag gcatcaaata
      901 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
      961 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatatacta
     1021 gtagcggccg ctgcagtccg gcaaaaaagg gcaaggtgtc accaccctgc cctttttctt
     1081 taaaaccgaa aagattactt cgcgttatgc aggcttcctc gctcactgac tcgctgcgct
     1141 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaatc tgttgtttgt
     1201 cggtgaacgc tctcctgagt aggacaaatc cgccgcccta gacctagggt acgggttttg
     1261 ctgcccgcaa acgggctgtt ctggtgttgc tagtttgtta tcagaatcgc agatccggct
     1321 tcagccggtt tgccggctga aagcactatt tcttccagaa ttgccatgat tttttcccca
     1381 cgggaggcgt cactggctcc cgtgttgtcg gcagctttga ttcgataagc agcatcgcct
     1441 gtttcaggct gtctatgtgt gactgttgag ctgtaacaag ttgtctcagg tgttcaattt
     1501 catgttctag ttgctttgtt ttactggttt cacctgttct attaggtgtt acatgctgtt
     1561 catctgttac attgtcgatc tgttcatggt gaacagcttt gaatgcacca aaaactcgta
     1621 aaagctctga tgtatctatc ttttttacac cgttttcatc tgtgcatatg gacagttttc
     1681 cctttgatat gtaacggtga acagttgttc tacttttgtt tgttagtctt gatgcttcac
     1741 tgatagatac aagagccata agaacctcag atccttccgt atttagccag tatgttctct
     1801 agtgtggttc gttgtttttg cgtgagccat gagaacgaac cattgagatc atacttactt
     1861 tgcatgtcac tcaaaaattt tgcctcaaaa ctggtgagct gaatttttgc agttaaagca
     1921 tcgtgtagtg tttttcttag tccgttatgt aggtaggaat ctgatgtaat ggttgttggt
     1981 attttgtcac cattcatttt tatctggttg ttctcaagtt cggttacgag atccatttgt
     2041 ctatctagtt caacttggaa aatcaacgta tcagtcgggc ggcctcgctt atcaaccacc
     2101 aatttcatat tgctgtaagt gtttaaatct ttacttattg gtttcaaaac ccattggtta
     2161 agccttttaa actcatggta gttattttca agcattaaca tgaacttaaa ttcatcaagg
     2221 ctaatctcta tatttgcctt gtgagttttc ttttgtgtta gttcttttaa taaccactca
     2281 taaatcctca tagagtattt gttttcaaaa gacttaacat gttccagatt atattttatg
     2341 aattttttta actggaaaag ataaggcaat atctcttcac taaaaactaa ttctaatttt
     2401 tcgcttgaga acttggcata gtttgtccac tggaaaatct caaagccttt aaccaaagga
     2461 ttcctgattt ccacagttct cgtcatcagc tctctggttg ctttagctaa tacaccataa
     2521 gcattttccc tactgatgtt catcatctga gcgtattggt tataagtgaa cgataccgtc
     2581 cgttctttcc ttgtagggtt ttcaatcgtg gggttgagta gtgccacaca gcataaaatt
     2641 agcttggttt catgctccgt taagtcatag cgactaatcg ctagttcatt tgctttgaaa
     2701 acaactaatt cagacataca tctcaattgg tctaggtgat tttaatcact ataccaattg
     2761 agatgggcta gtcaatgata attactagtc cttttccttt gagttgtggg tatctgtaaa
     2821 ttctgctaga cctttgctgg aaaacttgta aattctgcta gaccctctgt aaattccgct
     2881 agacctttgt gtgttttttt tgtttatatt caagtggtta taatttatag aataaagaaa
     2941 gaataaaaaa agataaaaag aatagatccc agccctgtgt ataactcact actttagtca
     3001 gttccgcagt attacaaaag gatgtcgcaa acgctgtttg ctcctctaca aaacagacct
     3061 taaaacccta aaggcttaag tagcaccctc gcaagctcgg gcaaatcgct gaatattcct
     3121 tttgtctccg accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc
     3181 gctcacggct ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca
     3241 tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat
     3301 ggcgggtctg ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga
     3361 ttttccagtc tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac
     3421 ccagtaaggc agcggtatca tcaacaggct tacccgtctt actgtcccta gtgcttggat
     3481 tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc agatggagtt
     3541 ctgaggtcat tactggatct atcaacagga gtccaagcga gctctcgaac cccagagtcc
     3601 cgcttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3661 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3721 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3781 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3841 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3901 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3961 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4021 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4081 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4141 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4201 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4261 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4321 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4381 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4441 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatcg
     4501 cgaaacgatc ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt
     4561 ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc
     4621 ccagctggca attccgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     4681 aggcgtatca cgaggccctt tcgtcttcac ctcgagtgcc acctgacgtc taagaaacca
     4741 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcagaat ttcagataaa
     4801 aaaaatcctt agctttcgct aaggatgatt tctggaattc gcggccgctt ctagag
//
