LOCUS       Exported                8206 bp ds-DNA     circular SYN 30-NOV-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pCali2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8206)
  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..8206
                     /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
                     /note="TlpA36"
                     /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"
     promoter        complement(5749..5778)
                     /note="pRM Promoter"
     misc_feature    5749..5765
                     /note="Operator OR3"
                     /note="J02459"
     misc_feature    5772..5788
                     /note="Operator OR2"
                     /note="J02459"
     promoter        5779..5837
                     /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    5796..5812
                     /note="Operator OR1"
                     /note="J02459"
     misc_feature    5967..5983
                     /note="Operator OL3"
                     /note="J02459"
     misc_feature    5987..6003
                     /note="Operator OL2"
                     /note="J02459"
     promoter        5994..6044
                     /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    6011..6027
                     /note="Operator OL1"
                     /note="J02459"
     RBS             6159..6166
                     /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             6173..6883
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /note="mCherry"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     promoter        6937..7013
                     /gene="lacI"
                     /note="lacI promoter"
     RBS             7021..7026
                     /note="ribosome binding site"
     CDS             7035..7748
                     /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      7857..7904
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
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 cgttgcgcta tcaccgcaag
     5761 ggataaatat ctaacaccgt gcgtgttgac tattttacct ctggcggtga taatggttgc
     5821 atgtactaag gaggttgtat ggaacaacgc ataaccctga aagattatgc aatgcgcttt
     5881 gggcaaacca agacagctaa aagatctctc acctaccaaa caatgccccc ctgcaaaaaa
     5941 taaattcata taaaaaacat acagataacc atctgcggtg ataaattatc tctggcggtg
     6001 ttgacataaa taccactggc ggtgatactg agcacatcag caggacgcac tgaccaccat
     6061 gaaggtgacg ctcttaaaaa ttaagccctg aagaagggca gcattcaaag cagaaggctt
     6121 tggggtgtgt gatacgaaac gaagcattgg ttaaaaatta aggagggaat tcatggtgag
     6181 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     6241 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     6301 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
     6361 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     6421 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     6481 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     6541 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     6601 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     6661 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     6721 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     6781 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     6841 gggccgccac tccaccggcg gcatggacga gctgtacaag taaaagcttg cggccgcata
     6901 atgcttaagt cgaacagaaa gtaatcgtat gagctcgaca ccatcgaatg gcgcaaacct
     6961 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtataag
     7021 aaggagatat acatatgagc acaaaaaaga aaccattaac acaagagcag cttgaggacg
     7081 cacgtcgcct taaagcaatt tatgaaaaaa agaaaaatga acttggctta tcccaggaat
     7141 ctgtcgcaga caagatgggg atggggcagt caggcgttgg tgctttattt aatggcatca
     7201 atgcattaaa tgcttataac gccgcattgc ttacaaaaat tctcaaagtt agcgttgaag
     7261 aatttagccc ttcaatcgcc agagaaatct acgagatgta tgaagcggtt agtatgcagc
     7321 cgtcacttag aagtgagtat gagtaccctg ttttttctca tgttcaggca gggatgttct
     7381 cacctgagct tagaaccttt accaaaggtg atgcggagag atgggtaagc acaaccaaaa
     7441 aagccagtga ttctgcattc tggcttgagg ttgaaggtaa ttccatgacc gcaccaacag
     7501 gctccaagcc aagctttcct gacggaatgt taattctcgt tgaccctgag caggctgttg
     7561 agccaggtga tttctgcata gccagacttg ggggtgatga gtttaccttc aagaaactga
     7621 tcagggatag cggtcaggtg tttttacaac cactaaaccc acagtaccca atgatcccat
     7681 gcaatgagag ttgttccgtt gtggggaaag ttatcgctag tcagtggcct gaagagacgt
     7741 ttggctgact cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg
     7801 actcgtctac tagcgcagct taattaacct aggctgctgc caccgctgag caataactag
     7861 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     7921 tatccggatt ggcgaatggg cgggatctcg acgctctccc ttatgcgact cctgcattag
     7981 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     8041 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     8101 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     8161 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacg
//
