LOCUS       Exported                7073 bp ds-DNA     circular SYN 30-NOV-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pTcI-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(139..168)
                     /note="pRM Promoter"
     misc_feature    139..155
                     /note="Operator OR3"
                     /note="J02459"
     misc_feature    162..178
                     /note="Operator OR2"
                     /note="J02459"
     promoter        169..227
                     /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    186..202
                     /note="Operator OR1"
                     /note="J02459"
     misc_feature    357..373
                     /note="Operator OL3"
                     /note="J02459"
     misc_feature    377..393
                     /note="Operator OL2"
                     /note="J02459"
     promoter        384..434
                     /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    401..417
                     /note="Operator OL1"
                     /note="J02459"
     RBS             549..556
                     /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             563..565
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     CDS             575..592
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             605..1315
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /note="mWasabi"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYKG"
     promoter        1372..1448
                     /gene="lacI"
                     /note="lacI promoter"
     RBS             1456..1461
                     /note="ribosome binding site"
     CDS             1470..2183
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /note="TcI"
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     terminator      2292..2339
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2376..2831
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2946..3806)
                     /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(3807..3899)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      3980..4568
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4998..5189)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5761..6843)
                     /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(6844..6921)
                     /gene="lacI"
                     /note="lacI promoter"
ORIGIN
        1 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
       61 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
      121 ccggcgtaga ggatcgagta tcaccgcaag ggataaatat ctaacaccgt gcgtgttgac
      181 tattttacct ctggcggtga taatggttgc atgtactaag gaggttgtat ggaacaacgc
      241 ataaccctga aagattatgc aatgcgcttt gggcaaacca agacagctaa aagatctctc
      301 acctaccaaa caatgccccc ctgcaaaaaa taaattcata taaaaaacat acagataacc
      361 atctgcggtg ataaattatc tctggcggtg ttgacataaa taccactggc ggtgatactg
      421 agcacatcag caggacgcac tgaccaccat gaaggtgacg ctcttaaaaa ttaagccctg
      481 aagaagggca gcattcaaag cagaaggctt tggggtgtgt gatacgaaac gaagcattgg
      541 ttaaaaatta aggagggaat tcatgggcag cagccatcac catcatcacc acagccagga
      601 tccaatggtg agcaagggcg aggagaccac aatgggcgta atcaagcccg acatgaagat
      661 caagctgaag atggagggca acgtgaatgg ccacgccttc gtgatcgagg gcgagggcga
      721 gggcaagccc tacgacggca ccaacaccat caacctggag gtgaaggagg gagcccccct
      781 gcccttctcc tacgacattc tgaccaccgc gttcagttac ggcaacaggg ccttcaccaa
      841 gtaccccgac gacatcccca actacttcaa gcagtccttc cccgagggct actcttggga
      901 gcgcaccatg accttcgagg acaagggcat cgtgaaggtg aagtccgaca tctccatgga
      961 ggaggactcc ttcatctacg agatacacct caagggcgag aacttccccc ccaacggccc
     1021 cgtgatgcag aaggagacca ccggctggga cgcctccacc gagaggatgt acgtgcgcga
     1081 cggcgtgctg aagggcgacg tcaagatgaa gctgctgctg gagggcggcg gccaccaccg
     1141 cgttgacttc aagaccatct acagggccaa gaaggcggtg aagctgcccg actatcactt
     1201 tgtggaccac cgcatcgaga tcctgaacca cgacaaggac tacaacaagg tgaccgttta
     1261 cgagatcgcc gtggcccgca actccaccga cggcatggac gagctgtaca agggctgaaa
     1321 gcttgcggcc gcataatgct taagtcgaac agaaagtaat cgtatgagct cgacaccatc
     1381 gaatggcgca aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     1441 tggtgaatgt ataagaagga gatatacata tgagcacaaa aaagaaacca ttaacacaag
     1501 agcagcttga ggacgcacgt cgccttaaag caatttatga aaaaaagaaa aatgaacttg
     1561 gcttatccca ggaatctgtc gcagacaaga tggggatggg gcagtcaggc gttggtgctt
     1621 tatttaatgg catcaatgca ttaaatgctt ataacgccgc attgcttaca aaaattctca
     1681 aagttagcgt tgaagaattt agcccttcaa tcgccagaga aatctacgag atgtatgaag
     1741 cggttagtat gcagccgtca cttagaagtg agtatgagta ccctgttttt tctcatgttc
     1801 aggcagggat gttctcacct gagcttagaa cctttaccaa aggtgatgcg gagagatggg
     1861 taagcacaac caaaaaagcc agtgattctg cattctggct tgaggttgaa ggtaattcca
     1921 tgaccgcacc aacaggctcc aagccaagct ttcctgacgg aatgttaatt ctcgttgacc
     1981 ctgagcaggc tgttgagcca ggtgatttct gcatagccag acttgggggt gatgagttta
     2041 ccttcaagaa actgatcagg gatagcggtc aggtgttttt acaaccacta aacccacagt
     2101 acccaatgat cccatgcaat gagagttgtt ccgttgtggg gaaagttatc gctagtcagt
     2161 ggcctgaaga gacgtttggc tgactcgagt ctggtaaaga aaccgctgct gcgaaatttg
     2221 aacgccagca catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg
     2281 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     2341 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     2401 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2461 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2521 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2581 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2641 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2701 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2761 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2821 gtttacaatt tctggcggca cgatggcatg agattatcaa aaaggatctt cacctagatc
     2881 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2941 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3001 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3061 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3121 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3181 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3241 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3301 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3361 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3421 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3481 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3541 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3601 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3661 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3721 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3781 gcgacacgga aatgttgaat actcatactc ttcctttttc aatcatgatt gaagcattta
     3841 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3901 aggtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3961 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4021 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4081 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4141 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4201 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4261 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4321 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4381 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4441 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4501 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4561 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4621 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4681 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4741 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4801 gcatatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4861 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4921 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4981 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5041 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     5101 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     5161 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     5221 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     5281 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     5341 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     5401 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     5461 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     5521 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     5581 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     5641 atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5701 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     5761 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5821 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5881 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     5941 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     6001 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     6061 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     6121 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     6181 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     6241 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     6301 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     6361 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     6421 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     6481 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     6541 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6601 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     6661 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     6721 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     6781 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     6841 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     6901 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     6961 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     7021 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata ccc
//
