LOCUS       Exported                5901 bp ds-DNA     circular SYN 30-NOV-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pLacI265-Wasabi
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5901)
  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..5901
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        35..115
                     /note="T7A1O4/O3 Promoter"
                     /note="Extrapolated from: 
                     
                     Lanzer, M., & Bujard, H. (1988). Promoters largely 
                     determine the efficiency of repressor action. Proceedings 
                     of the National Academy of Sciences of the United States of
                     America, 85(23), 8973–8977. 
                     http://doi.org/10.1073/pnas.85.23.8973
                     
                     Verified from: Lutz, R., & Bujard, H. (1997). Independent 
                     and tight regulation of transcriptional units in 
                     escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 
                     regulatory elements. Nucleic Acids Research, 25(6), 
                     1203–1210. doi:10.1093/nar/25.6.1203"
     protein_bind    56..72
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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)."
     protein_bind    89..105
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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)."
     RBS             127..132
     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..897
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /note="mWasabi"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYKG"
     terminator      1006..1053
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1090..1545
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(1660..2520)
                     /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(2521..2613)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      2694..3282
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3468..3610
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(3712..3903)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4475..5557)
                     /codon_start=1
                     /product="Temperature-sensitive LacI
                     Leakage 35-39
                     Transition 39-40"
                     /note="LacIts(G265D)"
                     /note="Chao, Y. P., Chern, J. T., Wen, C. S., & Fu, H. 
                     (2002). Construction and characterization of 
                     thermo-inducible vectors derived from heat-sensitive lacl 
                     genes in combination with the T7 A1 promoter. Biotechnology
                     and Bioengineering, 79(1), 1–8. 
                     http://doi.org/10.1002/bit.10304"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVDADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5558..5635)
                     /gene="lacI"
                     /note="lacI promoter"
ORIGIN
        1 atgcgtccgg cgtagaggat cgagctcgag aaaatttatc aaaaagagtg ttgacttgtg
       61 agcggataac aatgatactt agattcaatt gtgagcggat aacaatttca cacagaattc
      121 attaaagagg agaaattaac 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 gggctgactc
      901 gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact
      961 agcgcagctt aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt
     1021 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1081 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1141 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1201 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1261 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1321 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1381 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1441 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1501 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttctggc ggcacgatgg
     1561 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1621 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1681 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1741 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1801 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1861 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1921 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1981 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2041 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     2101 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     2161 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     2221 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     2281 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     2341 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     2401 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     2461 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2521 actcttcctt tttcaatcat gattgaagca tttatcaggg ttattgtctc atgagcggat
     2581 acatatttga atgtatttag aaaaataaac aaataggtca tgaccaaaat cccttaacgt
     2641 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2701 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2761 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2821 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2881 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2941 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3001 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3061 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3121 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3181 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3241 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3301 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3361 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3421 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3481 tttctcctta cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa
     3541 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3601 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3661 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3721 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     3781 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     3841 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     3901 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     3961 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4021 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4081 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4141 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4201 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4261 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4321 ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca
     4381 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     4441 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4501 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4561 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     4621 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     4681 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     4741 atcccactac cgagatgtcc gcatcaacgc gcagcccgga ctcggtaatg gcgcgcattg
     4801 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     4861 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     4921 tcggctgaat ttgattgcga gtgagatatt tatgccaacc agccagacgc agacgcgccg
     4981 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     5041 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     5101 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     5161 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5221 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5281 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     5341 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     5401 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     5461 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     5521 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     5581 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     5641 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     5701 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     5761 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     5821 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     5881 gcgccggtga tgccggccac g
//
