LOCUS       Exported                7074 bp ds-DNA     circular SYN 30-NOV-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pTcI38-Wasabi
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7074)
  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..7074
                     /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..1449
                     /gene="lacI"
                     /note="lacI promoter"
     RBS             1457..1462
                     /note="ribosome binding site"
     CDS             1471..2184
                     /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="VSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAASLTRILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMLSPELRTFTKGGAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGGEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     misc_feature    1471..1473
                     /note="M1V"
                     /note="cI 7-2"
     misc_feature    1618..1620
                     /note="A50 (Synonymous)"
                     /note="cI 7-2"
     misc_feature    1663..1665
                     /note="L65S"
                     /note="cI 7-2"
     misc_feature    1672..1674
                     /note="K68R"
                     /note="cI 7-2"
     misc_feature    1813..1815
                     /note="F115L"
                     /note="cI 7-2"
     misc_feature    1846..1848
                     /note="D126G"
                     /note="cI 7-2"
     misc_feature    1852..1854
                     /note="E128 (Synonymous)"
                     /note="cI 7-2"
     misc_feature    1855..1857
                     /product="cI 7-2"
                     /note="R129 (Synonymous)"
     misc_feature    1924..1926
                     /note="T152 (Synonymous)"
                     /note="cI 7-2"
     misc_feature    2023..2025
                     /note="L185 (Synonymous)"
                     /note="cI 7-2"
     misc_feature    2032..2034
                     /note="D188G"
                     /note="cI 7-2"
     terminator      2293..2340
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2377..2832
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2947..3807)
                     /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(3808..3900)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      3981..4569
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4999..5190)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5762..6844)
                     /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(6845..6922)
                     /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 aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     1441 gtggtgaatg tataagaagg agatatacat gtgagcacaa aaaagaaacc attaacacaa
     1501 gagcagcttg aggacgcacg tcgccttaaa gcaatttatg aaaaaaagaa aaatgaactt
     1561 ggcttatccc aggaatctgt cgcagacaag atggggatgg ggcagtcagg cgttggtgcc
     1621 ttatttaatg gcatcaatgc attaaatgct tataacgccg catcgcttac aagaattctc
     1681 aaagttagcg ttgaagaatt tagcccttca atcgccagag aaatctacga gatgtatgaa
     1741 gcggttagta tgcagccgtc acttagaagt gagtatgagt accctgtttt ttctcatgtt
     1801 caggcaggga tgctctcacc tgagcttaga acctttacca aaggtggtgc ggaaaggtgg
     1861 gtaagcacaa ccaaaaaagc cagtgattct gcattctggc ttgaggttga aggtaattcc
     1921 atgacagcac caacaggctc caagccaagc tttcctgacg gaatgttaat tctcgttgac
     1981 cctgagcagg ctgttgagcc aggtgatttc tgcatagcca gactcggggg tggtgagttt
     2041 accttcaaga aactgatcag ggatagcggt caggtgtttt tacaaccact aaacccacag
     2101 tacccaatga tcccatgcaa tgagagttgt tccgttgtgg ggaaagttat cgctagtcag
     2161 tggcctgaag agacgtttgg ctgactcgag tctggtaaag aaaccgctgc tgcgaaattt
     2221 gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc tgctgccacc
     2281 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2341 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     2401 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2461 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2521 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2581 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2641 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2701 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2761 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2821 cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct tcacctagat
     2881 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     2941 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3001 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3061 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3121 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3181 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3241 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3301 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3361 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3421 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3481 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3541 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3601 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3661 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3721 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3781 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat tgaagcattt
     3841 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3901 taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3961 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4021 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4081 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4141 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4201 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4261 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4321 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4381 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4441 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4501 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4561 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4621 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4681 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4741 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4801 cgcatatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4861 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     4921 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4981 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     5041 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     5101 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5161 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5221 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     5281 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5341 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     5401 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     5461 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     5521 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     5581 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     5641 gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5701 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5761 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5821 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     5881 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     5941 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6001 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     6061 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6121 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6181 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6241 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6301 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6361 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6421 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6481 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6541 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6601 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6661 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6721 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6781 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6841 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     6901 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     6961 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     7021 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat accc
//
