LOCUS       62056_10355        7784 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7784)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7784
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     protein_bind    166..290
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(334..636)
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             complement(981..1637)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(1691..1721)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     protein_bind    complement(1746..1870)
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1886..2539)
                     /codon_start=1
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             complement(2558..2575)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     RBS             complement(2588..2610)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(2625..2649)
                     /label=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)."
     promoter        complement(2650..2668)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2977..3054
                     /label=lacI promoter
     CDS             3055..4134
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    4150..4171
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             4946..5134
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    5239..5381
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5567..6155)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6329..7186)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7187..7291)
                     /label=AmpR promoter
     rep_origin      complement(7318..7773)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtcaccac tttgtacaag
      181 aaagctgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
      241 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gtcgacctgc
      301 agactggctg tgtataaggg agcctgacat ttatattccc cagaacatca ggttaatggc
      361 gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac
      421 cggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac
      481 cgggtaaagt tcacgggaga ctttatctga cagcagacgt gcactggcca gggggatcac
      541 catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg
      601 gctctctctt ttataggtgt aaaccttaaa ctgcatttca ccagtccctg ttctcgtcag
      661 caaaagagcc gttcatttca ataaaccggg cgacctcagc catcccttcc tgattttccg
      721 ctttccagcg ttcggcacgc agacgacggg cttcattctg catggttgtg cttaccagac
      781 cggagatatt gacatcatat atgccttgag caactgatag ctgtcgctgt caactgtcac
      841 tgtaatacgc tgcttcatag cacacctctt tttgacatac ttcgggtata catatcagta
      901 tatattctta taccgcaaaa atcagcgcgc aaatacgcat actgttatct ggcttttagt
      961 aagccggatc cacgcgttta cgccccgccc tgccactcat cgcagtactg ttgtaattca
     1021 ttaagcattc tgccgacatg gaagccatca cagacggcat gatgaacctg aatcgccagc
     1081 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     1141 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     1201 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     1261 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     1321 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     1381 tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc
     1441 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     1501 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     1561 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     1621 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgccgg atcctaactc
     1681 aaaatccaca cattatacga gccggaagca taaagtgtaa agcctggggt gcctaatgcg
     1741 gccgccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca
     1801 aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca gcttttttgt
     1861 acaaacttgt catggtacca tccgattttg gaggatggtc gccaccacca aacgtggctt
     1921 gccagccctg caaaggccat gctatatact tgctggattt caagtactta tcaatttgtg
     1981 ggatagcttc aatacgtttt ttaaaacaaa ctaattttgg gaacgcatcc aggcacattg
     2041 ggtccatgta taaaacaaca tcaagagcgt catacaacat gaagtcagga tgggttacat
     2101 gatcaccatt taaatatgtt ttatgacata aacgatcttc gaacattttc agcatttcag
     2161 gtagcttgct aagaaaatca actttgagag tttcaaagtc tttactatat gcaattctcg
     2221 aaacaccgta tctaatatcc aaaaccgctc cttcaagcat tgaaatctct gcacgctctt
     2281 ttggacaacc acccaacatg ttgtgcttgt cagctatata acgtatgatg gccatagact
     2341 gtgttaattt aacatcacca tcaatataat aaggaagatt gggaaactcc aaacccaatt
     2401 caaacttttt gtttcgccat ttatcacctt catcgcgctc atacaaatgc tcttcatatt
     2461 tttcttcaag atattccaaa agaagtcgag tgggttgcac aaggccctta attttccaat
     2521 aacctagtat aggggacatg gaattgctac tagtgttgtg atggtgatgg tgatgtttca
     2581 tggtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct
     2641 cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg
     2701 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     2761 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     2821 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     2881 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     2941 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     3001 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     3061 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     3121 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     3181 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     3241 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     3301 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     3361 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     3421 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     3481 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     3541 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     3601 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     3661 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     3721 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     3781 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     3841 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     3901 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     3961 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     4021 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     4081 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     4141 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
     4201 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     4261 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
     4321 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
     4381 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
     4441 cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct
     4501 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     4561 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     4621 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     4681 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     4741 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     4801 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     4861 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     4921 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     4981 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     5041 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
     5101 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5161 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5221 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     5281 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat atgcggtgtg
     5341 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     5401 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5461 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5521 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5581 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5641 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5701 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5761 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5821 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5881 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5941 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6001 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6061 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6121 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6181 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6241 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6301 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6361 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6421 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6481 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6541 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6601 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac
     6661 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6721 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6781 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6841 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6901 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6961 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7021 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7081 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7141 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7201 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7261 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaaa
     7321 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     7381 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     7441 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     7501 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     7561 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     7621 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     7681 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     7741 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gcca
//
