LOCUS       Exported                7073 bp ds-DNA     circular SYN 30-NOV-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pTcI42-Wasabi
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7073)
  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..7073
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(25..54)
                     /note="pRM Promoter"
     misc_feature    25..41
                     /note="Operator OR3"
                     /note="J02459"
     misc_feature    48..64
                     /note="Operator OR2"
                     /note="J02459"
     promoter        55..113
                     /note="pR promoter"
                     /note="Kincade, J. M., & Pieter, L. (1991). Bacteriophage 
                     lambda promoters pL and pR: sequence determinants of in 
                     vivo activity and of sensitivity to the DNA gyrase 
                     inhibitor, coumermycin. Gene, 97, 7–12."
     misc_feature    72..88
                     /note="Operator OR1"
                     /note="J02459"
     misc_feature    243..259
                     /note="Operator OL3"
                     /note="J02459"
     misc_feature    263..279
                     /note="Operator OL2"
                     /note="J02459"
     promoter        270..320
                     /note="pL Promoter"
                     /note="Kincade, J. M., & Pieter, L. (1991). Bacteriophage 
                     lambda promoters pL and pR: sequence determinants of in 
                     vivo activity and of sensitivity to the DNA gyrase 
                     inhibitor, coumermycin. Gene, 97, 7–12."
     misc_feature    287..303
                     /note="Operator OL1"
                     /note="J02459"
     RBS             435..442
                     /note="Elvin, C. M., Thompson, P. R., Argall, M. E., 
                     Hendry, P., Stamford, N. P., Lilley, P. E., & Dixon, N. E. 
                     (1990). Modified bacteriophage lambda promoter vectors for 
                     overproduction of proteins in Escherichia coli. Gene, 
                     87(1), 123–126. 
                     http://doi.org/10.1016/0378-1119(90)90503-J"
     CDS             449..451
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     CDS             461..478
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             491..1201
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /note="mWasabi"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYKG"
     promoter        1258..1334
                     /gene="lacI"
                     /note="lacI promoter"
     RBS             1342..1347
                     /note="ribosome binding site"
     CDS             1356..2069
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /note="lambda repressor (ts)"
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKNPLTQEQLEDARRLKAIYEKKKNELGLTQESVADKMGMGQ
                     SGVGALFNGINALNAHNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPEPRTCTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     misc_feature    1371..1373
                     /note="K6N"
                     /note="cI 5-4"
     misc_feature    1452..1454
                     /note="S33T"
                     /note="cI 5-4"
     misc_feature    1506
                     /note="L51 (Synonymous)"
                     /note="cI 5-4"
     misc_feature    1536..1538
                     /note="Y61H"
                     /note="cI Mutant 5-4"
     misc_feature    1710..1712
                     /note="L119P"
                     /note="cI 5-4 mutant"
     misc_feature    1719..1721
                     /product="cI 5-4 mutant"
                     /note="F122C"
     terminator      2178..2225
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2262..2717
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2832..3692)
                     /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(3693..3785)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      3866..4454
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4884..5075)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5647..6729)
                     /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(6730..6807)
                     /gene="lacI"
                     /note="lacI promoter"
ORIGIN
        1 atgcgtccgg cgtagaggat cgagtatcac cgcaagggat aaatatctaa caccgtgcgt
       61 gttgactatt ttacctctgg cggtgataat ggttgcatgt actaaggagg ttgtatggaa
      121 caacgcataa ccctgaaaga ttatgcaatg cgctttgggc aaaccaagac agctaaaaga
      181 tctctcacct accaaacaat gcccccctgc aaaaaataaa ttcatataaa aaacatacag
      241 ataaccatct gcggtgataa attatctctg gcggtgttga cataaatacc actggcggtg
      301 atactgagca catcagcagg acgcactgac caccatgaag gtgacgctct taaaaattaa
      361 gccctgaaga agggcagcat tcaaagcaga aggctttggg gtgtgtgata cgaaacgaag
      421 cattggttaa aaattaagga gggaattcat gggcagcagc catcaccatc atcaccacag
      481 ccaggatcca atggtgagca agggcgagga gaccacaatg ggcgtaatca agcccgacat
      541 gaagatcaag ctgaagatgg agggcaacgt gaatggccac gccttcgtga tcgagggcga
      601 gggcgagggc aagccctacg acggcaccaa caccatcaac ctggaggtga aggagggagc
      661 ccccctgccc ttctcctacg acattctgac caccgcgttc agttacggca acagggcctt
      721 caccaagtac cccgacgaca tccccaacta cttcaagcag tccttccccg agggctactc
      781 ttgggagcgc accatgacct tcgaggacaa gggcatcgtg aaggtgaagt ccgacatctc
      841 catggaggag gactccttca tctacgagat acacctcaag ggcgagaact tcccccccaa
      901 cggccccgtg atgcagaagg agaccaccgg ctgggacgcc tccaccgaga ggatgtacgt
      961 gcgcgacggc gtgctgaagg gcgacgtcaa gatgaagctg ctgctggagg gcggcggcca
     1021 ccaccgcgtt gacttcaaga ccatctacag ggccaagaag gcggtgaagc tgcccgacta
     1081 tcactttgtg gaccaccgca tcgagatcct gaaccacgac aaggactaca acaaggtgac
     1141 cgtttacgag atcgccgtgg cccgcaactc caccgacggc atggacgagc tgtacaaggg
     1201 ctgaaagctt gcggccgcat aatgcttaag tcgaacagaa agtaatcgta tgagctcgac
     1261 accatcgaat ggcgcaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     1321 tcagggtggt gaatgtataa gaaggagata tacatatgag cacaaaaaag aatccattaa
     1381 cacaagagca gcttgaggac gcacgtcgcc ttaaagcaat ttatgaaaaa aagaaaaatg
     1441 aacttggctt aacccaggaa tctgtcgcag acaagatggg gatggggcag tcaggcgttg
     1501 gtgctctatt taatggcatc aatgcattaa atgctcataa cgccgcattg cttacaaaaa
     1561 ttctcaaagt tagcgttgaa gaatttagcc cttcaatcgc cagagaaatc tacgagatgt
     1621 atgaagcggt tagtatgcag ccgtcactta gaagtgagta tgagtaccct gttttttctc
     1681 atgttcaggc agggatgttc tcacctgagc ctagaacctg taccaaaggt gatgcggaga
     1741 gatgggtaag cacaaccaaa aaagccagtg attctgcatt ctggcttgag gttgaaggta
     1801 attccatgac cgcaccaaca ggctccaagc caagctttcc tgacggaatg ttaattctcg
     1861 ttgaccctga gcaggctgtt gagccaggtg atttctgcat agccagactt gggggtgatg
     1921 agtttacctt caagaaactg atcagggata gcggtcaggt gtttttacaa ccactaaacc
     1981 cacagtaccc aatgatccca tgcaatgaga gttgttccgt tgtggggaaa gttatcgcta
     2041 gtcagtggcc tgaagagacg tttggctgac tcgagtctgg taaagaaacc gctgctgcga
     2101 aatttgaacg ccagcacatg gactcgtcta ctagcgcagc ttaattaacc taggctgctg
     2161 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     2221 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     2281 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2341 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2401 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2461 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2521 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2581 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2641 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2701 attaacgttt acaatttctg gcggcacgat ggcatgagat tatcaaaaag gatcttcacc
     2761 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2821 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2881 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2941 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3001 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3061 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3121 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3181 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3241 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3301 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3361 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     3421 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     3481 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3541 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3601 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     3661 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatc atgattgaag
     3721 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3781 acaaataggt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3841 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3901 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3961 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4021 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4081 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4141 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4201 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4261 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4321 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4381 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4441 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4501 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4561 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4621 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4681 cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4741 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     4801 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4861 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     4921 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     4981 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     5041 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     5101 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     5161 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     5221 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     5281 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     5341 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     5401 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     5461 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     5521 agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5581 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     5641 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5701 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     5761 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     5821 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     5881 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac
     5941 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6001 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6061 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6121 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     6181 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     6241 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6301 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6361 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6421 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6481 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6541 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6601 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6661 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6721 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     6781 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     6841 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     6901 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     6961 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     7021 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acg
//
