LOCUS       sequence-130223-        5562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of mouse RALA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5562)
  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 5562)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2013.04"; journalName: "Cell"; date: "2013-05-23- 
            23"; volume: "153"; issue: "5"; pages: "1050-63"
COMMENT     Sequence Label: RALA
FEATURES             Location/Qualifiers
     source          1..5562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..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(1612..1634)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1779..1797)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1865..1969
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1970..2038,2039..2830)
                     /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(2188..2207)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3001..3589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3490..3509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3743..3760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3833..3910
                     /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             3911..4993
                     /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(3930..3949)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5006..5027
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5042..5047,5048..5065,5066..5072)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5080..5096
                     /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     5104..5120
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5104..5120
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5116..5562,1..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(5136..5153)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5136..5152)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5145..5167)
                     /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 aaacaagccc aagggtcaga
      961 attctttggc cttacacaaa gtcatcatgg tgggcagtgg tggtgtgggc aagtctgccc
     1021 tgactctgca gttcatgtac gacgagtttg tagaggacta tgaacctacc aaagcggaca
     1081 gctacaggaa gaaggtagtc ctggacgggg aagaagtgca gatcgacatc ttagatacag
     1141 cggggcagga ggactatgct gcaattagag acaactactt ccgaagtggg gagggattcc
     1201 tctgtgtctt ctctatcaca gagatggagt ccttcgcagc taccgcggac ttcagggagc
     1261 agattttaag agtaaaagaa gatgagaatg ttccatttct cctggttggt aacaagtcag
     1321 atctagaaga taaaaggcag gtttctgtag aagaggcaaa aaacagagct gaccagtgga
     1381 acgttaacta tgtggagacg tctgctaaaa cgcgcgccaa cgttgacaag gtattttttg
     1441 atttaatgag ggaaatacga gccagaaaga tggaagacag caaagaaaaa aatggaaaaa
     1501 agaagaggaa aagtttagcc aagagaatca gagaaagatg ctgcatttta taacaattca
     1561 tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     1621 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     1681 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     1741 tagcgatagc ggagtgtata attcttgaag acgaaagggc ctcgtgatac gcctattttt
     1801 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     1861 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1921 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1981 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2041 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2101 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2161 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     2221 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2281 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2341 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2401 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2461 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2521 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2581 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2641 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2701 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2761 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2821 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2881 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2941 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3001 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3061 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataaatt ccgacaccat
     3841 cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     3901 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     3961 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     4021 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     4081 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     4141 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     4201 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     4261 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     4321 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     4381 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     4441 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     4501 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     4561 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     4621 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     4681 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     4741 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     4801 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     4861 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     4921 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     4981 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     5041 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     5101 acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt
     5161 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     5221 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     5281 aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg
     5341 gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt
     5401 tacgatgcgc ccatctacac caacgtaacc tatcccatta cggtcaatcc gccgtttgtt
     5461 cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta
     5521 caggaaggcc agacgcgaat tatttttgat ggcgttggaa tt
//
