LOCUS       Exported                6486 bp ds-DNA     circular SYN 30-NOV-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pTetR89-Wasabi
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6486)
  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..6486
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(25..648)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR
                     Temperature sensitive from 28C to 42C
                     Wissmann, A., Wray, L. V., Somaggio, U., Baumeister, R., 
                     Geissendorfer, M., & Hillen, W. (1991). Selection for Tn10 
                     tet repressor binding to tet operator in Escherichia coli: 
                     Isolation of temperature-sensitive mutants and 
                     combinatorial mutagenesis in the DNA binding motif. 
                     Genetics, 128(2), 225–232."
                     /note="tsTetR AD89"
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCDLLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     promoter        664..719
                     /gene="tetR"
                     /note="tetR/tetA promoters"
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    670..688
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    700..718
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             727..729
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     CDS             739..756
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             769..1479
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /note="mWasabi"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYKG"
     terminator      1591..1638
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1675..2130
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2245..3105)
                     /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(3106..3198)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      3279..3867
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4297..4488)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5060..6142)
                     /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(6143..6220)
                     /gene="lacI"
                     /note="lacI promoter"
ORIGIN
        1 atgcgtccgg cgtagaggat cgagtcagga cccactttca catttaagtt gtttttctaa
       61 tccgcatatg atcaattcaa ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa
      121 taattcgata gcttgtcgta ataatggcgg catactatca gtagtaggtg tttccctttc
      181 ttctttagcg acttgatgct cttgatcttc caatacgcaa cctaaagtaa aatgccccac
      241 agcgctgagt gcatataatg cattctctag tgaaaaacct tgttggcata aaaaggctaa
      301 ttgattttcg agagtttcat actgtttttc tgtaggccgt gtacctaaat gtacttttgc
      361 tccatcgcga tgacttagta aatcacatct aaaactttta gcgttattac gtaaaaaatc
      421 ttgccagctt tccccttcta aagggcaaaa gtgagtatgg tgcctatcta acatctcaat
      481 ggctaaggcg tcgagcaaag cccgcttatt ttttacatgc caatacaatg taggctgctc
      541 tacacctagc ttctgggcga gtttacgggt tgttaaacct tcgattccga cctcattaag
      601 cagctctaat gcgctgttaa tcactttact tttatctaat ctagacatta attcctaatt
      661 tttgttgaca ctctatcatt gatagagtta ttttaccact ccctatcagt gatagagaaa
      721 agtgaaatgg gcagcagcca tcaccatcat caccacagcc aggatccaat ggtgagcaag
      781 ggcgaggaga ccacaatggg cgtaatcaag cccgacatga agatcaagct gaagatggag
      841 ggcaacgtga atggccacgc cttcgtgatc gagggcgagg gcgagggcaa gccctacgac
      901 ggcaccaaca ccatcaacct ggaggtgaag gagggagccc ccctgccctt ctcctacgac
      961 attctgacca ccgcgttcag ttacggcaac agggccttca ccaagtaccc cgacgacatc
     1021 cccaactact tcaagcagtc cttccccgag ggctactctt gggagcgcac catgaccttc
     1081 gaggacaagg gcatcgtgaa ggtgaagtcc gacatctcca tggaggagga ctccttcatc
     1141 tacgagatac acctcaaggg cgagaacttc ccccccaacg gccccgtgat gcagaaggag
     1201 accaccggct gggacgcctc caccgagagg atgtacgtgc gcgacggcgt gctgaagggc
     1261 gacgtcaaga tgaagctgct gctggagggc ggcggccacc accgcgttga cttcaagacc
     1321 atctacaggg ccaagaaggc ggtgaagctg cccgactatc actttgtgga ccaccgcatc
     1381 gagatcctga accacgacaa ggactacaac aaggtgaccg tttacgagat cgccgtggcc
     1441 cgcaactcca ccgacggcat ggacgagctg tacaagggct gactcgagtc tggtaaagaa
     1501 accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta
     1561 acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1621 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1681 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1741 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1801 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1861 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1921 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1981 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2041 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2101 attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa
     2161 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2221 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2281 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2341 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     2401 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2461 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2521 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     2581 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2641 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2701 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     2761 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     2821 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     2881 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     2941 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3001 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3061 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3121 atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3181 tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3241 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3301 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3361 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3421 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3481 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3541 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3601 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3661 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3721 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3781 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3841 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3901 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3961 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4021 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4081 ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg
     4141 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4201 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4261 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4321 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     4381 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     4441 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     4501 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     4561 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     4621 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4681 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4741 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4801 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     4861 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     4921 cctcaacgac aggagcacga tcatgctagt catgccccgc gcccaccgga aggagctgac
     4981 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5041 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5101 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5161 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5221 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5281 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5341 tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5401 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5461 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5521 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5581 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5641 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5701 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5761 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     5821 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     5881 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     5941 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     6001 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     6061 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6121 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     6181 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6241 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6301 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6361 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6421 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6481 gccacg
//
