LOCUS       40924_44247        6747 bp DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6747)
  AUTHORS   Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B, Hahn 
            KM
  TITLE     A genetically encoded photoactivatable Rac controls the motility of 
            living cells.
  JOURNAL   Nature. 2009 Sep 3. 461(7260):104-8.
  PUBMED    19693014
REFERENCE   2  (bases 1 to 6747)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6747)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2009 Sep 3. 461(7260):104-8."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     complement(1..706)
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     terminator      complement(719..766)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     836..855
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also
                     called rb-glob-pA-term-R"
     polyA_signal    complement(854..909)
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     955..974
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(1064..1087)
                     /codon_start=1
                     /label=8xHis
                     /note="8xHis affinity tag"
                     /translation="HHHHHHHH"
     CDS             complement(1094..1126)
                     /codon_start=1
                     /label=HSV tag
                     /note="HSV (herpes simplex virus) epitope tag"
                     /translation="QPELAPEDPED"
     CDS             complement(1166..1738)
                     /codon_start=1
                     /label=Rac1 (Q61L)
                     /note="constitutively active mutant of human Rac family
                     small GTPase 1"
                     /translation="ELIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMV
                     DGKPVNLGLWDTAGLEDYDRLRPLSYPQTDVFLICFSLVSPASFHHVRAKWYPEVRHHC
                     PNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQRG
                     LKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     CDS             complement(2168..2872)
                     /codon_start=1
                     /label=mCherry
                     /note="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     CDS             complement(2879..2896)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     promoter        complement(2917..3026)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     protein_bind    complement(3042..3066)
                     /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(3067..3085)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3127..3146)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     primer_bind     complement(3208..3230)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(3436..3635)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(3637..3940)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_recomb     complement(4008..4835)
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             5078..5935
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5951..6538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    6706..6739
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 aattccaaaa agctcaacta caaatttgat cgcggacgtg ttagccgaca caattaatag
       61 gcgtcgtgtg gctatggcaa aatcgtcttc ggaagcaact tctaacgacg agggttggga
      121 cgacgacgat aatcggccta ataaagctaa cacgcccgat gttaaatatg tccaagctct
      181 atttaacgtg tttacgtcga gtcaattgta cactaacgac agtgatgaaa gaaatacaaa
      241 agcgcataat attttgaacg acgtcgaacc tttattacaa aacaaaacac aaacgaatat
      301 cgacaaagct agattgctgc tacaagattt ggcaagtttt gtggcgttga gcgaaaatcc
      361 attagatagt ccagccatcg gttcggaaaa acaacccttg tttgaaacta atcgaaacct
      421 attttacaaa tctattgagg atttaatatt taaattcaga tataaagacg ctgaaaatca
      481 tttgattttc gctctaacat accaccctaa agattataaa tttaatgaat tattaaaata
      541 cgtacaacaa ttgtctgtaa atcaacaacg cacagaatct agcgcttaat aaatgtacta
      601 ataacaatgt atcgtgtttt aatacgccgg accagtgaac agaggtgcgt ctggtgcaaa
      661 ctcctttact ttgaacacca gggaaacttc aaggagaatt tcctccgcat gctttcagca
      721 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttac atatgggcat
      781 atgttgccaa actctaaacc aaatactcat tctgatgttt taaatgattt gccctcccat
      841 atgtccttcc gagtgagaga cacaaaaaat tccaacacac tattgcaatg aaaataaatt
      901 tcctttatta gccagaagtc agatgctcaa ggggcttcat gatgtcccca taatttttgg
      961 cagagggaaa aagatcgatc tcagtggtat ttgtgagcca gggcattggc cacaccagcc
     1021 accaccttct gataggcagc ctgcacctga ggttaatcac ttagtgatgg tgatggtgat
     1081 ggtggtgctc gagatcctcg gggtcttccg gggcgagttc tggctggctt gcacgtgtat
     1141 acagctgtgc ggccgcaagc ttttacaaca gcaggcattt tctcttcctc ttcttcacgg
     1201 gaggcgggca gaggactgct cggatcgctt cgtcaaacac tgtcttgagg cctcgctgtg
     1261 tgagcgccga gcactccagg tattttacag caccaatctc cttagccatg gctagaccct
     1321 gcggataggt gatgggagtc agcttcttct ccttcagttt ctcgatcgtg tctttatcat
     1381 ccctaagatc aagtttagtt cccactagga tgatgggagt gttgggacag tggtgccgca
     1441 cctcaggata ccactttgca cggacgtggt ggaatgatgc aggactcaca agggaaaagc
     1501 aaattaagaa cacatctgtt tgcggatagg atagggggcg taatctgtca taatcttcta
     1561 gtccagctgt atcccataag cccagattca ccggttttcc atctaccata acattggcag
     1621 aataattgtc aaagacagta gggatatatt ctccaggaaa tgcattggtt gtgtaactga
     1681 tcagtaggca agttttacct acagctccgt ctcccaccac cacacacttg ataagttctt
     1741 ttgccgcctc atcaatattt tctgcagttt tcttaatcag catgactccc tctctctcgg
     1801 cagcatctcg gacatgctca gttccatcca actgaacccc aataaagtac tggacatctc
     1861 ccttctgatc tcgcataggc tgcaagtgaa agaggttcca gaacttttta ccactctttg
     1921 tataattaat cagctgaaca gtgacctctg tttggttatc tatggcatct ctaatttttc
     1981 tcactgtcgc gcgatcagtt tcaggacctt gtagaaacct gcagtttctt cccaaaattt
     2041 cttcacggct atattctgtc aactgcaaga aactatcgga cgcgaatata atgggattat
     2101 ctggcaatct tgggtcagta atgacaaagt tcttctcaat acgttcaagt gtagtagcca
     2161 aggatccctt gtacagctcg tccatgccgc cggtggagtg gcggccctcg gcgcgttcgt
     2221 actgttccac gatggtgtag tcctcgttgt gggaggtgat gtccaacttg atgttgacgt
     2281 tgtaggcgcc gggcagctgc acgggcttct tggccttgta ggtggtcttg acctcagcgt
     2341 cgtagtggcc gccgtccttc agcttcagcc tctgcttgat ctcgcccttc agggcgccgt
     2401 cctcggggta catccgctcg gaggaggcct cccagcccat ggtcttcttc tgcattacgg
     2461 ggccgtcgga ggggaagttg gtgccgcgca gcttcacctt gtagatgaac tcgccgtcct
     2521 gcagggagga gtcctgggtc acggtcacca cgccgccgtc ctcgaagttc atcacgcgct
     2581 cccacttgaa gccctcgggg aaggacagct tcaagtagtc ggggatgtcg gcggggtgct
     2641 tcacgtaggc cttggagccg tacatgaact gaggggacag gatgtcccag gcgaagggca
     2701 gggggccacc cttggtcacc ttcagcttgg cggtctgggt gccctcgtag gggcggccct
     2761 cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga gccctccatg tgcaccttga
     2821 agcgcatgaa ctccttgatg atggccatgt tatcctcctc gcccttgctc acagatccgt
     2881 gatggtggtg atggtgtgcc atggtatatc tcctttgatt gtaaataaaa tgtaatttac
     2941 agtatagtat tttaattaat atacaaatga tttgataata attcttattt aactataata
     3001 tattgtgttg ggttgaatta aaggtcccgg attaactccg gggaattgtt atccgctcac
     3061 aattccccta tagtgagtcg tattaatttc ggtccgatcc aattctttgc caaaatgatg
     3121 agacagcaca ataaccagca cgttgcccag gagctgtagg aaaaagaaga aggcatgaac
     3181 atggttagca gaggctctag agccgccggt cacacgccag aagccgaacc ccgccctgcc
     3241 ccgtcccccc cgaaggcagc cgtccccccg cggacagccc cgagccgctc cccccgcaca
     3301 aagggcccga tacttacctg cccagtgcct cacgaccaac ttctgcacgt taagttcgag
     3361 actggcgtta gcaatttaac tgtgataaac taccgcaata aagcatctag tgatctgacg
     3421 gttcactaaa cgacgtctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg
     3481 cgtcaacggg gcggggttat tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa
     3541 caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta
     3601 tccacgccca ttggtgtact gccaaaaccg catcagcatg gtaatagcga tgactaatac
     3661 gtagatgtac tgccaagtag gaaagtcccg taaggtcatg tactgggcat aatgccaggc
     3721 gggccattta ccgtcattga cgtcaatagg gggcggactt ggcatatgat acacttgatg
     3781 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     3841 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     3901 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     3961 atgaactaat gaccccgtaa ttgattacta ttaataggcc ggccatcagc aactatatat
     4021 tgatagacat ttccagtttg tgatattagt ttgtgcgtct cattacaatg gctgttattt
     4081 ttaacaacaa acaactgctc gcagacaata gtatagaaaa gggaggtgaa ctgtttttgt
     4141 ttaacggttc gtacaacatt ttggaaagtt atgttaatcc ggtgctgcta aaaaatggtg
     4201 taattgaact agaagaagct gcgtactatg ccggcaacat attgtacaaa accgacgatc
     4261 ccaaattcat tgattatata aatttaataa ttaaagcaac acactccgaa gaactaccag
     4321 aaaatagcac tgttgtaaat tacagaaaaa ctatgcgcag cggtactata caccccatta
     4381 aaaaagacat atatatttat gacaacaaaa aatttactct atacgataga tacatatatg
     4441 gatacgataa taactatgtt aatttttatg aagagaaaaa tgaaaaagag aaggaatacg
     4501 aagaagaaga cgacaaggcg tctagtttat gtgaaaataa aattatattg tcgcaaatta
     4561 actgtgaatc atttgaaaat gattttaaat attacctcag cgattataac tacgcgtttt
     4621 caattataga taatactaca aatgttcttg ttgcgtttgg tttgtatcgt taataaaaaa
     4681 caaatttgac atttataatt gttttattat tcaataatta caaataggat tgagaccctt
     4741 gcagttgcca gcaaacggac agagcttgtc gacagttttc attttttggc agtttggtgg
     4801 tttatacaca tcttgggatt tgattaaaag atgcatgact tcacccacac acttggagtc
     4861 gtggttgtag aaatgcgtca gagccttgat taaacatcgg ttttcacact tggcacacga
     4921 aaccacgttt gcaatgtaac agtttagtat aaacctcttc ctgtacatac cggcgcgttt
     4981 atacacatct tgggatttga ttaaaagatg caggaaacgc gcggacatga gacaataacc
     5041 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5101 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     5161 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     5221 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5281 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5341 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5401 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5461 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     5521 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5581 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     5641 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5701 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5761 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5821 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5881 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaaca
     5941 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6001 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6061 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6121 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6181 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6241 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6301 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6361 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6421 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6481 acctctgact tgagcgtcga tttttgtgat gctcgtcagg gggcggagcc tatggaaaaa
     6541 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6601 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6661 taccgctcgc cgcagccgaa cgaccgagcg cagcgttccg agcttataac ttcgtataat
     6721 gtatgctata cgaagttatg cgatcgc
//
