LOCUS       40924_43278        5150 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_41, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5150)
  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 5150)
  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 5150)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5150)
  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..5150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..31
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    complement(37..56)
                     /label=lac operator (symmetric)
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     misc_RNA        73..123
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      147..165
                     /note="RBS32A; derived from BBa_B0032"
                     /regulatory_class="ribosome_binding_site"
     CDS             166..1059
                     /codon_start=1
                     /product="RepA"
                     /label=RepA
                     /note="ColE2 replication protein"
                     /protein_id="AVR55117.1"
                     /translation="MSAVLQRFREKLPHKPYCTNDFAYGVRILPKNIAILARFIQQNQP
                     HALYWLPFDVDRTGASIDWSDRNCPAPNITVKNPRNGHAHLLYALALPVRTAPDASASA
                     LRYAAAIERALCEKLGADVNYSGLICKNPCHPEWQEVEWREEPYTLDELADYLDLSASA
                     RRSVDKNYGLGRNYHLFEKVRKWAYRAIRQGWPVFSQWLDAVIQRVEMYNASLPVPLSP
                     AECRAIGKSIAKYTHRKFSPEGFSAVQAARGRKGGTKSKRAAVPTSARSLKPWEALGIS
                     RATYYRKLKCDPDLAK"
     terminator      1073..1144
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      1160..1187
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1194..1214
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1215..1272
                     /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."
     CDS             complement(1433..2221)
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     rep_origin      complement(2816..3360)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     terminator      complement(3850..3893)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(3939..5018)
                     /label=lacI
                     /note="lac repressor"
     regulatory      complement(5019..5100)
                     /label=LacI promoter
                     /note="LacI promoter"
                     /regulatory_class="promoter"
ORIGIN
        1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattctat
       61 ggactatgtt tagctgtcac cggatgtgct ttccggtctg atgagtccgt gaggacgaaa
      121 cagcctctac aaataatttt gtttaatcac acaggaaagt actaaatgag cgccgtactt
      181 caacgcttcc gtgaaaaatt accgcacaaa ccgtactgta ctaacgattt cgcgtacggc
      241 gttcgcattc tgccgaaaaa cattgccatt cttgcccgtt tcatccagca gaaccagcca
      301 catgcactgt actggcttcc ctttgacgtg gaccggacgg gggcatcaat cgactggagc
      361 gaccggaatt gtccggcccc gaacatcacc gtaaaaaatc cccgtaacgg gcacgcgcat
      421 ctgctctacg cgctcgccct tcctgtgaga actgcgccgg atgcatcggc ttcggcgctc
      481 agatacgctg ccgctattga gcgtgcgttg tgtgaaaaac tgggcgcgga tgtgaattac
      541 agcggcctga tctgcaaaaa tccgtgccac cctgaatggc aggaagtgga atggcgcgag
      601 gaaccctaca ctctcgacga actggctgat tatctggatc tgagcgcctc agcgcgccgt
      661 agcgtcgata aaaattacgg gctggggcga aactaccatc tgttcgaaaa ggtccgtaaa
      721 tgggcctaca gagcgattcg tcagggctgg cctgtattct cacaatggct tgatgccgtt
      781 atccagcgtg tcgaaatgta caacgcttcg cttcccgttc cgctttctcc ggctgaatgt
      841 cgggctattg gcaagagcat tgcgaaatat acacacagga aattctcacc agagggattt
      901 tccgctgtac aggccgctcg cggtcgcaag ggcggaacta aatctaagcg cgcagcagtt
      961 cctacatcag cacgttcgct gaaaccgtgg gaggcattag gcatcagtcg agcgacgtac
     1021 taccgaaaat taaaatgtga cccagacctc gcaaaataat aggtccaggc atcaaataaa
     1081 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     1141 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt atatactagt
     1201 agcggccgct gcagtccggc aaaaaagggc aaggtgtcac caccctgccc tttttcttta
     1261 aaaccgaaaa gattacttcg cgttatgcag gcttcctcgc tcactgactc gctgcgctcg
     1321 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatctc gagtcccgtc
     1381 aagtcagcgt aatgctctgc cagtgttaca accaattaac caattctgat tatttgccga
     1441 ctaccttggt gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgtgcgcg
     1501 aggccaagcg atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct
     1561 gatactgggc cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt
     1621 tgccggttac tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc
     1681 cagcccagtc gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat
     1741 cctgttcagg aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat
     1801 gttctcttgc ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga
     1861 tacctgcaag aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat
     1921 aacgccacgg aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct
     1981 cgctctctac aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg
     2041 tttcatcaag ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc
     2101 cgccatccac tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct
     2161 cgatgacgcc aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca
     2221 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat
     2281 ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt tgttgaataa
     2341 atcgaacttt tgctgagttg aaggatcaga tcacgcatct tcccgacaac gcagaccgtt
     2401 ccgtggcaaa gcaaaagttc aaaatcacca actggtccac ctacaacaaa gctctcatca
     2461 accgtggctc cctcactttc tggctggatg atggggcgat tcaggcctgg tatgagtcag
     2521 caacaccttc ttcacgaggc agacctcagc gctagcggag tgtatactgg cttactatgt
     2581 tggcactgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg
     2641 gtgcgtcagc agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta
     2701 cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg
     2761 gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc
     2821 ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa
     2881 acccgacagg actataaaga taccaggcgt ttcccctggc ggctccctcg tgcgctctcc
     2941 tgttcctgcc tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc
     3001 cacgcctgac actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac
     3061 cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3121 aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc
     3181 ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct
     3241 ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc
     3301 tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa
     3361 gaagatcatc ttattaaggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3421 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3481 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3541 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3601 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3661 ccgcgatacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3721 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat tccatggtgc
     3781 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     3841 cgaggcagaa tttcagataa aaaaaatcct tagctttcgc taaggatgat ttctggtcgg
     3901 ccaattcgcg ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3961 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4021 ttgggcgcca gggtggtttt tcttttcacc agtgagactg gcaacagctg attgcccttc
     4081 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4141 aaatcctgtt tgatggtggt taacggcggg atataacatg agctatcttc ggtatcgtcg
     4201 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4261 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4321 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4381 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4441 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4501 tgctccacgc ccagtcgcgt accgtcctca tgggagaaaa taatactgtt gatgggtgtc
     4561 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4621 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4681 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4741 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4801 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4861 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4921 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     4981 gcatactctg cgacatcgta taacgttact ggtttcatat tcaccaccct gaattgactc
     5041 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggcgcgccgc
     5101 ttcgtcaggc cacatagctt tcttgttctg atcggaacga tcgttggctg
//
