LOCUS       Exported                7175 bp ds-DNA     circular SYN 30-NOV-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pTlpA36-Wasabi
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7175)
  AUTHORS   Dan Piraner
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Nov 30, 2016 from SnapGene 3.2.1
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7175
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        47..98
                     /bound_moiety="TlpA"
                     /note="TlpA Promoter"
     RBS             129..134
                     /note="ribosome binding site"
     CDS             142..144
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     CDS             154..171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             184..894
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /note="mWasabi"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYKG"
     promoter        951..1028
                     /gene="lacI"
                     /note="lacI promoter"
     RBS             1036..1041
                     /note="ribosome binding site"
     CDS             1050..2165
                     /codon_start=1
                     /product="Evolved TlpA
                     Expression starts at ~34C
                     Expression midpoint at ~37C
                     Expression max at ~43C
                     Refer to data from September 30 2015
                     
                     Mutations from wt: 
                     P60L, D135V, K187R, K202I, L208Q
                     
                     Formerly known as TlpA Mutant 30"
                     /note="mTlpA1"
                     /translation="MRPATYEPEQIIEAGLALQAEGRNITGFALRNQVGGGNPTRLRQI
                     WDEYQASQSTVVTELVAELPVEVAEEVKAVSAALSERITQLATELNDKAVRAAERRVAE
                     VTRAAGEQTAQAERELADAAQTVDDLEEKLVELQDRYDSLTLALESERSLRQQHDVEMA
                     QLKERLAAAEENTRQREERYQEQRTVLQDALNAEQAQHINTREDQQKRLEQISAEANAR
                     TEELKSERDKVNTLLTRLESQENALASERQQHLATRETLQQRLEQAIADTQARAGEIAL
                     ERDRVSSLTARLESQEKASSEQLVRMGSEIASLTERCTQLENQRDDARLETMGEKETVA
                     ALRGEAEALKRQNQSLMAALSGNKQTGGQNA"
     misc_feature    1228
                     /note="P60L"
                     /note="TlpA 36 mutation"
     misc_feature    1453
                     /note="D135V"
                     /note="TlpA 36 Mutation"
     misc_feature    1609
                     /note="K187R"
                     /note="TlpA 36 Mutation"
     misc_feature    1654
                     /note="K202I"
                     /note="TlpA 36 Mutation"
     misc_feature    1672
                     /note="L208Q"
                     /note="TlpA 36 mutation"
     terminator      2280..2327
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2364..2819
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2934..3794)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(3795..3887)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      3968..4556
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4986..5177)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5749..6831)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="lacI"
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6832..6909)
                     /gene="lacI"
                     /note="lacI promoter"
ORIGIN
        1 atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaatttta atttgtttgt
       61 tagttagttt atttgttggt ttgtttgtgt tataatatcc tctagaaata attttgttta
      121 actttaagaa ggagatatac catgggcagc agccatcacc atcatcacca cagccaggat
      181 ccaatggtga gcaagggcga ggagaccaca atgggcgtaa tcaagcccga catgaagatc
      241 aagctgaaga tggagggcaa cgtgaatggc cacgccttcg tgatcgaggg cgagggcgag
      301 ggcaagccct acgacggcac caacaccatc aacctggagg tgaaggaggg agcccccctg
      361 cccttctcct acgacattct gaccaccgcg ttcagttacg gcaacagggc cttcaccaag
      421 taccccgacg acatccccaa ctacttcaag cagtccttcc ccgagggcta ctcttgggag
      481 cgcaccatga ccttcgagga caagggcatc gtgaaggtga agtccgacat ctccatggag
      541 gaggactcct tcatctacga gatacacctc aagggcgaga acttcccccc caacggcccc
      601 gtgatgcaga aggagaccac cggctgggac gcctccaccg agaggatgta cgtgcgcgac
      661 ggcgtgctga agggcgacgt caagatgaag ctgctgctgg agggcggcgg ccaccaccgc
      721 gttgacttca agaccatcta cagggccaag aaggcggtga agctgcccga ctatcacttt
      781 gtggaccacc gcatcgagat cctgaaccac gacaaggact acaacaaggt gaccgtttac
      841 gagatcgccg tggcccgcaa ctccaccgac ggcatggacg agctgtacaa gggctgaaag
      901 cttgcggccg cataatgctt aagtcgaaca gaaagtaatc gtatgagctc gacaccatcg
      961 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     1021 tggtgaatgt ataagaagga gatatacata tgcgtccggc gacatacgaa ccagaacaga
     1081 ttattgaagc agggctggcc ctgcaggctg aaggacggaa tatcaccggg ttcgcactac
     1141 gtaaccaggt gggtggcggc aatccgacac gtctccgcca gatatgggac gaataccagg
     1201 cttcacagag cacggtcgtc actgaactcg ttgccgagct gccagtggaa gtggctgaag
     1261 aagtgaaggc cgtctccgcc gcgctgtccg aacgcatcac ccagctggcg acagaactga
     1321 atgacaaggc ggtccgggct gcagaacgcc gggttgcgga agtcacgcgt gctgccggtg
     1381 aacagaccgc acaggcagag cgggagctgg ccgacgccgc gcagacagtc gacgacctgg
     1441 aagaaaaact ggttgaactg caggacagat atgacagttt gacgctggcg ctggagtcag
     1501 aacgttcact gcgtcagcag catgatgtgg agatggccca gctgaaagag cgtcttgcgg
     1561 ccgctgaaga gaatacccgt cagcgagagg aacggtatca ggagcagagg acagtgctgc
     1621 aggatgcgct taatgcggag caggcacagc acataaacac gcgggaagac cagcagaaac
     1681 gactggagca aatttctgcc gaagctaatg cgcgtacaga agaactgaag tctgaacgcg
     1741 ataaagtcaa tactctcctt acccgccttg aatcgcagga aaatgcgctg gcctcagaac
     1801 gtcagcagca tctggccacc cgcgaaacgc tgcagcaacg cctcgagcag gccatcgctg
     1861 acacgcaggc gcgcgccggt gagattgcac ttgaacgtga cagagtcagc agcctcaccg
     1921 caaggctgga atcgcaggaa aaggcctcct cggagcaact ggtgcgtatg ggcagtgaaa
     1981 tagccagtct gacagagcgt tgcacacagc tggaaaacca gcgtgatgat gcccgtctgg
     2041 agacgatggg ggagaaagaa acggtcgcgg cactgcgtgg tgaggctgaa gccctgaagc
     2101 gtcagaacca gtcactgatg gcggcgcttt caggcaataa acagaccggt ggccagaatg
     2161 cgtgaggtac cctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca
     2221 tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac
     2281 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2341 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2401 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2461 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2521 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2581 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2641 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2701 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2761 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     2821 tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2881 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2941 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3001 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3061 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3121 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3181 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3241 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3301 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3361 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3421 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3481 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3541 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3601 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3661 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3721 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3781 tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg
     3841 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca
     3901 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3961 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4021 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4081 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4141 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4201 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4261 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4321 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4381 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4441 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4501 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4561 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4621 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4681 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4741 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg
     4801 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4861 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4921 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4981 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     5041 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     5101 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     5161 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     5221 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     5281 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     5341 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     5401 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     5461 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     5521 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     5581 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc
     5641 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5701 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     5761 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5821 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     5881 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     5941 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     6001 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     6061 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     6121 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6181 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6241 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6301 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6361 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6421 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6481 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6541 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     6601 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6661 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6721 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     6781 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     6841 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     6901 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     6961 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     7021 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     7081 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     7141 caaccgcacc tgtggcgccg gtgatgccgg ccacg
//
