LOCUS       Exported                5523 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expression of mouse RhoA.
ACCESSION   .
VERSION     .
KEYWORDS    RhoA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5523)
  AUTHORS   Fritsch R, de Krijger I, Fritsch K, George R, Reason B, Kumar MS, 
            Diefenbacher M, Stamp G, Downward J
  TITLE     RAS and RHO families of GTPases directly regulate distinct 
            phosphoinositide 3-kinase isoforms.
  JOURNAL   Cell. 2013 May 23;153(5):1050-63. doi: 10.1016/j.cell.2013.04.031.
  PUBMED    23706742
REFERENCE   2  (bases 1 to 5523)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5523
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        183..211
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1573..1595)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1740..1758)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1826..1930
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1931..2791
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2149..2168)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2962..3550
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3451..3470
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3704..3721
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3794..3871
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3872..4954
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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"
     primer_bind     complement(3891..3910)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    4967..4988
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5003..5033
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5041..5057
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     primer_bind     5065..5081
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5065..5081
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5077..81
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5097..5114)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5097..5113)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5106..5128)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccgctgc catcaggaag aaactggtga
      961 ttgttggtga tggagcttgt ggtaagacat gcttgctcat agtcttcagc aaggaccagt
     1021 tcccagaggt ctatgtgccc acggtgtttg aaaactatgt ggcggatatc gaggtggatg
     1081 ggaagcaggt agagttggct ttatgggaca cagctggaca ggaagattat gaccgcctgc
     1141 ggcctctctc ttatccagac accgatgtta tattgatgtg tttttccatt gacagccctg
     1201 atagtttaga aaacatccca gaaaaatgga ctccagaagt caagcatttc tgtccaaatg
     1261 tgcccatcat cctggttggg aacaagaagg accttcggaa tgacgagcac acgagacggg
     1321 agttggccaa aatgaagcag gagccggtaa aacctgaaga aggcagagat atggcaaaca
     1381 ggattggcgc ttttgggtac atggagtgtt cagcaaagac caaagatgga gtgagagagg
     1441 tttttgagat ggccacgaga gctgctctgc aagctagacg tgggaagaaa aagtctgggt
     1501 gcctcatctt gtgagaattc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga
     1561 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     1621 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     1681 cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg
     1741 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     1801 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1861 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1921 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     1981 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2041 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2101 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2161 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2221 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2281 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2341 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2401 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2461 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2521 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     2581 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     2641 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     2701 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     2761 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2821 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     2881 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2941 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3001 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3061 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3121 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3181 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3241 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3301 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3361 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3421 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3481 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3541 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3601 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3661 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3721 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3781 ccgcataaat tccgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg
     3841 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     3901 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     3961 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     4021 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     4081 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     4141 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     4201 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     4261 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     4321 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     4381 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     4441 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     4501 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     4561 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     4621 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     4681 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     4741 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     4801 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     4861 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     4921 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     4981 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     5041 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac ggattcactg
     5101 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     5161 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     5221 tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa
     5281 gcggtgccgg aaagctggct ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc
     5341 tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtaac ctatcccatt
     5401 acggtcaatc cgccgtttgt tcccacggag aatccgacgg gttgttactc gctcacattt
     5461 aatgttgatg aaagctggct acaggaaggc cagacgcgaa ttatttttga tggcgttgga
     5521 att
//
