LOCUS       40924_43308        6629 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_56, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6629)
  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 6629)
  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 6629)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6629)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Biotechnol. (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (06-MAR-2018) Synthetic Biology Center, Department of Biological 
            Engineering, Massachusetts Institute of Technology, 77 Massachusetts
            Avenue, Cambridge, MA 02139, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..19
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase
                     recognition (Reznikoff et al., 2004)"
     terminator      complement(1114..1157)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    1160..1181
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with
                     'ATG'"
     regulatory      1194..1241
                     /note="PJ23105; derived from BBa_J23105"
                     /regulatory_class="promoter"
     misc_RNA        1243..1293
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      1317..1335
                     /note="B0032; derived from BBa_B0032"
                     /regulatory_class="ribosome_binding_site"
     CDS             1336..2049
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Nager et al., 2011)"
     terminator      2069..2140
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      2156..2183
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             2453..3241
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     misc_feature    complement(3337..3355)
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase
                     recognition (Reznikoff et al., 2004)"
     terminator      3366..3460
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3565..4992
                     /label=Tn5 transposase
                     /note="transposase from the bacterial Tn5 transposon
                     (Reznikoff, 1993)"
     promoter        5049..5140
                     /label=AmpR promoter
     CDS             5141..5797
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     terminator      5804..5843
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            5991..6099
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      6110..6498
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     terminator      complement(6529..6615)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
ORIGIN
        1 ctgtctctta tacacatctt tgtgtctcag gccgcctagg ccagacttaa gccagttcat
       61 tgcttcaacc gcagctacca ccgtcacgct cacagattcc caaatcagct tagccatgta
      121 tccagcagcc tgattcggct gagtgaacat cagacccttg ccggaatcaa tagctggctg
      181 aatggtatct tccagcactt gttgacggaa gccgaactct ttggacccgt aagccagcgt
      241 catgactgaa ctcttagtca cactgcgagt aacaccgtaa gccagccatt gaccagccag
      301 tgccttagtg cccagcttga ctttctcaga gatttcacca gtgttctcat cggtcacggt
      361 aactacttcg ttatcggtcc cattgattgc gtctgcttgt agaatctcgt tgactttctt
      421 agcaacaatc ccgtagatgt cctgaacggt ttcactagga agcaagttaa ccgcgcgacc
      481 acctacctca tctcggagca tcgcggagaa gtgctggatg ccagagcaag acccgtcaaa
      541 ttacgccagc ggaagggagc agttatagct caggccgtgg tgctgtaccc cagcgtactc
      601 aaagcagaac gcaaggaagc agaacggaga atcttgctca gcccaccaag tgttctccag
      661 tggagactta gcgcaagcca tgatgttctc gtggttttcc tcaatgaact tgatgcgctc
      721 agggaacgga accttgtcga cacccgcaca gtttgcaccg tggattttca gccagtagta
      781 accttcctta ccgattggtt tacctttcgc cagcgtaagc agtcctttgg tcatatcgtt
      841 accttgcggg ttgaacattg acacagcgta aacacgaccg cgccagtcca tgttgtaagg
      901 gaaccagatg gccttatggt tagcaaactt attggcttgc tcaagcatga actcaaggct
      961 gatacggcga gacttgcgag ccttgtcctt gcggtacaca gcagcggcag cacgtttcca
     1021 cgcggtgaga gcctcaggat ttgccacctg acgtctaaga aaccattatt atcatgacat
     1081 taacctataa aaataggcgt atcacgaggc agaatttcag ataaaaaaaa tccttagctt
     1141 tcgctaagga tgatttctgg aattcgcggc cgcttctaga gggaggtgtc ttcggcgcgc
     1201 ctttacggct agctcagtcc taggtactat gctagcaagg tagctgtcac cggatgtgct
     1261 ttccggtctg atgagtccgt gaggacgaaa cagcctctac aaataatttt gtttaatcac
     1321 acaggaaagt actagatgag caaaggagaa gaacttttca ctggagttgt cccaattctt
     1381 gttgaattag atggtgatgt taatgggcac aaattttctg tccgtggaga gggtgaaggt
     1441 gatgctacaa acggaaaact cacccttaaa tttatttgca ctactggaaa actacctgtt
     1501 ccgtggccaa cacttgtcac tactctgacc tatggtgttc aatgcttttc ccgttatccg
     1561 gatcacatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa
     1621 cgcactatat ctttcaaaga tgacgggacc tacaagacgc gtgctgaagt caagtttgaa
     1681 ggtgataccc ttgttaatcg tatcgagtta aagggtattg attttaaaga agatggaaac
     1741 attcttggac acaaactcga gtacaacttt aactcacaca atgtatacat cacggcagac
     1801 aaacaaaaga atggaatcaa agctaacttc aaaattcgcc acaacgttga agatggttcc
     1861 gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta
     1921 ccagacaacc attacctgtc gacacaatct gtcctttcga aagatcccaa cgaaaagcgt
     1981 gaccacatgg tccttcttga gtttgtaact gctgctggga ttacacatgg catggatgag
     2041 ctctacaaat aatactagag ccaggcatca aataaaacga aaggctcagt cgaaagactg
     2101 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
     2161 accttcgggt gggcctttct gcgtttatag aagacatcgc ttactagtag cggccgccaa
     2221 agcccgccga aaggcgggct tttctgtatt taaatgaacc ttgaccgaac gcagcggtgg
     2281 taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg tacagtctat
     2341 gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag
     2401 cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca tcatgaggga
     2461 agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca
     2521 tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa
     2581 gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg
     2641 gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct
     2701 ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc
     2761 taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga
     2821 gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt
     2881 tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt
     2941 tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga
     3001 gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc
     3061 gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt
     3121 catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc
     3181 agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa
     3241 ataagacaat tgtctaatta attgcggacc ctagaggtcc ccttttttat tttaaaaatt
     3301 ttttcacaaa acggtttaca agcataaaat ctctgaagat gtgtataaga gacagactag
     3361 tcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag
     3421 aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaggggtcc cctggtttaa
     3481 actacacaag tagcgtcctg aacggaacct ttcccgtttt ccagaatctg atgttccatg
     3541 tgacctccta acatggtaac gttcatgatt accagtgcac tgcatcgtgc ggcggattgg
     3601 gcgaaaagcg tgttttctag tgctgcgctg ggtgatccgc gtcgtaccgc gcgtctggtg
     3661 aatgttgcgg cgcaactggc caaatatagc ggcaaaagca ttaccattag cagcgaaggc
     3721 agcaaagcca tgcaggaagg cgcgtatcgt tttattcgta atccgaacgt gagcgcggaa
     3781 gcgattcgta aagcgggtgc catgcagacc gtgaaactgg cccaggaatt tccggaactg
     3841 ctggcaattg aagataccac ctctctgagc tatcgtcatc aggtggcgga agaactgggc
     3901 aaactgggta gcattcagga taaaagccgt ggttggtggg tgcatagcgt gctgctgctg
     3961 gaagcgacca cctttcgtac cgtgggcctg ctgcatcaag aatggtggat gcgtccggat
     4021 gatccggcgg atgcggatga aaaagaaagc ggcaaatggc tggccgctgc tgcaacttcg
     4081 cgtctgagaa tgggcagcat gatgagcaac gtgattgcgg tgtgcgatcg tgaagcggat
     4141 attcatgcgt atctgcaaga taaactggcc cataacgaac gttttgtggt gcgtagcaaa
     4201 catccgcgta aagatgtgga aagcggcctg tatctgtatg atcacctgaa aaaccagccg
     4261 gaactgggcg gctatcagat tagcattccg cagaaaggcg tggtggataa acgtggcaaa
     4321 cgtaaaaacc gtccggcgcg taaagcgagc ctgagcctgc gtagcggccg tattaccctg
     4381 aaacagggca acattaccct gaacgcggtg ctggccgaag aaattaatcc gccgaaaggc
     4441 gaaaccccgc tgaaatggct gctgctgacc agcgagccgg tggaaagtct ggcccaagcg
     4501 ctgcgtgtga ttgatattta tacccatcgt tggcgcattg aagaatttca caaagcgtgg
     4561 aaaacgggtg cgggtgcgga acgtcagcgt atggaagaac cggataacct ggaacgtatg
     4621 gtgagcattc tgagctttgt ggcggtgcgt ctgctgcaac tgcgtgaatc ttttactccg
     4681 ccgcaagcac tgcgtgcgca gggcctgctg aaagaagcgg aacacgttga aagccagagc
     4741 gcggaaaccg tgctgacccc ggatgaatgc caactgctgg gctatctgga taaaggcaaa
     4801 cgcaaacgca aagaaaaagc gggcagcctg caatgggcgt atatggcgat tgcgcgtctg
     4861 ggcggcttta tggatagcaa acgtaccggc attgcgagct ggggtgcgct gtgggaaggt
     4921 tgggaagcgc tgcaaagcaa actggatggc tttctggccg cgaaagacct gatggcgcag
     4981 ggcattaaaa tctaatcggc gatcgctagt agcccgccta atgagcgggc ttttttttaa
     5041 ttcccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5101 accctgataa atgcttcaat aatattgaaa aaggaagagt atggagaaaa aaatcactgg
     5161 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
     5221 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
     5281 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
     5341 gaatgctcat ccggaatttc gtatggcaat gaaagacggt gagctggtga tatgggatag
     5401 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
     5461 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
     5521 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
     5581 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
     5641 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
     5701 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
     5761 attacaacag tactgcgatg agtggcaggg cggggcgtaa accgatacaa ttaaaggctc
     5821 cttttggagc cttttttttt ggacacgcgt ttgtcctttt ccgctgcata accctgcttc
     5881 ggggtcatta tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt
     5941 tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata
     6001 ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg
     6061 gcggtgctca acgggaatcc tgctctgcga ggctggccgt aggccggccg atctgaagat
     6121 cagcagttca acctgttgat agtacgtact aagctctcat gtttcacgta ctaagctctc
     6181 atgtttaacg tactaagctc tcatgtttaa cgaactaaac cctcatggct aacgtactaa
     6241 gctctcatgg ctaacgtact aagctctcat gtttcacgta ctaagctctc atgtttgaac
     6301 aataaaatta atataaatca gcaacttaaa tagcctctaa ggttttaagt tttataagaa
     6361 aaaaaagaat atataaggct tttaaagcct ttaaggttta acggttgtgg acaacaagcc
     6421 agggatgtaa cgcactgaga agcccttaga gcctctcaaa gcaattttga gtgacacagg
     6481 aacacttaac ggctgacatg gggcgcgccc agctgtctag ggcggcggat ttgtcctact
     6541 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
     6601 ctttcgtttt atttgatgcc tttaattaa
//
