LOCUS       sequence-238117-        6342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of RALA S157A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6342)
  AUTHORS   Hiatt SM, Neu MB, Ramaker RC, Hardigan AA, Prokop JW, Hancarova M, 
            Prchalova D, Havlovicova M, Prchal J, Stranecky V, Yim DKC, Powis Z,
            Keren B, Nava C, Mignot C, Rio M, Revah-Politi A, Hemati P, Stong N,
            Iglesias AD, Suchy SF, Willaert R, Wentzensen IM, Wheeler PG, Brick 
            L, Kozenko M, Hurst ACE, Wheless JW, Lacassie Y, Myers RM, Barsh GS,
            Sedlacek Z, Cooper GM
  TITLE     De novo mutations in the GTP/GDP-binding region of RALA, a RAS-like 
            small GTPase, cause intellectual disability and developmental delay.
  JOURNAL   PLoS Genet. 2018 Nov 30;14(11):e1007671. doi: 
            10.1371/journal.pgen.1007671. eCollection 2018 Nov.
  PUBMED    30500825
REFERENCE   2  (bases 1 to 6342)
  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"; journalName: "PLoS 
            Genet."; date: "2018-11-30"; volume: "14(11):e1007671. doi"; pages: 
            " 10.1371/journal.pgen.1007671. eCollection 2018 Nov"
COMMENT     Sequence Label: RALA S157A
FEATURES             Location/Qualifiers
     source          1..6342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             22..39
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(725..743)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      739..786
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1024..1042)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1110..1214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1215..1283,1284..2075)
                     /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(1433..1452)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2246..2834
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2735..2754
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2988..3005
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3020..3159
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3085..3104)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3245..3267
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3261..3452)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4764..5846)
                     /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     5808..5827
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5847..5924)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6130..6149
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6233..6252
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6233..6251
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6252..6276
                     /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             6325..6342
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gtgaattcgc tcggttccgg gcttgtaccg cgcggatctc atatgggatc ccacatggct
       61 gcaaataagc ccaagggtca gaattctttg gctttacaca aagtcatcat ggtgggcagt
      121 ggtggcgtgg gcaagtcagc tctgactcta cagttcatgt acgatgagtt tgtggaggac
      181 tatgagccta ccaaagcaga cagctatcgg aagaaggtag tgctagatgg ggaggaagtc
      241 cagatcgata tcttagatac agctgggcag gaggactacg ctgcaattag agacaactac
      301 ttccgaagtg gggaggggtt cctctgtgtt ttctctatta cagaaatgga atcctttgca
      361 gctacagctg acttcaggga gcagatttta agagtaaaag aagatgagaa tgttccattt
      421 ctactggttg gtaacaaatc agatttagaa gataaaagac aggtttctgt agaagaggca
      481 aaaaacagag ctgagcagtg gaatgttaac tacgtggaaa cagctgctaa aacacgagct
      541 aatgttgaca aggtattttt tgatttaatg agagaaattc gagcgagaaa gatggaagac
      601 agcaaagaaa agaatggaaa aaagaagagg aaaagtttag ccaagagaat cagagaaaga
      661 tgctgcattt tataagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      721 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      781 tttttgctga aaggaggaac tatatccgga tatcccgcaa gaggcccggc agtaccggca
      841 taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat
      901 tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc
      961 attaaagcta gcttatcgat gataagctgt caaacatgag aattaattct tgaagacgaa
     1021 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     1081 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1141 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1201 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1261 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1321 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1381 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1441 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1501 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1561 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1621 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1681 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1741 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     1801 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1861 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1921 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1981 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2041 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2101 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2161 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2221 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2281 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2341 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2401 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2461 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2521 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2581 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2641 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2701 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2761 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2821 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2881 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2941 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3001 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3061 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3121 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3181 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3241 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3301 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3361 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3421 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3481 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3541 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3601 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     3661 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     3721 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     3781 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     3841 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     3901 acgatcatgc gcacccgtgg ccaggaccca acgctgcccg agatgcgccg cgtgcggctg
     3961 ctggagatgg cggacgcgat ggatatgttc tgccaagggt tggtttgcgc attcacagtt
     4021 ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc gaggtgccgc
     4081 cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc
     4141 agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg
     4201 cggcataaat cgccgtgacg atcagcggtc caatgatcga agttaggctg gtaagagccg
     4261 cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac agcatggcct
     4321 gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg aaggccatcc
     4381 agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc atgccggcga
     4441 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4501 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4561 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4621 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4681 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4741 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4801 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4861 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4921 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4981 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     5041 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5101 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5161 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5221 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5281 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5341 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5401 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5461 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5521 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5581 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5641 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5701 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5761 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5821 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5881 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5941 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     6001 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6061 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6121 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6181 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
     6241 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaaa
     6301 ctttaagaag gagatataca tatgcatcat catcatcatc ac
//
