LOCUS       sequence-238116-        6342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of RALA V25M.
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 V25M
FEATURES             Location/Qualifiers
     source          1..6342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             40..57
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(743..761)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      757..804
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1042..1060)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1128..1232
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1233..1301,1302..2093)
                     /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(1451..1470)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2264..2852
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2753..2772
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3006..3023
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3038..3177
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3103..3122)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3263..3285
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3279..3470)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4782..5864)
                     /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     5826..5845
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5865..5942)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6148..6167
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6251..6270
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6251..6269
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6270..6294
                     /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)."
ORIGIN
        1 catcatcatc atcatcacgt gaattcgctc agttccgggc ttgtaccgcg cggatctcat
       61 atgggatccc acatggctgc aaataagccc aagggtcaga attctttggc tttacacaaa
      121 gtcatcatgg tgggcagtgg tggcatgggc aagtcagctc tgactctaca gttcatgtac
      181 gatgagtttg tggaggacta tgagcctacc aaagcagaca gctatcggaa gaaggtagtg
      241 ctagatgggg aggaagtcca gatcgatatc ttagatacag ctgggcagga ggactacgct
      301 gcaattagag acaactactt ccgaagtggg gaggggttcc tctgtgtttt ctctattaca
      361 gaaatggaat cctttgcagc tacagctgac ttcagggagc agattttaag agtaaaagaa
      421 gatgagaatg ttccatttct actggttggt aacaaatcag atttagaaga taaaagacag
      481 gtttctgtag aagaggcaaa aaacagagct gagcagtgga atgttaacta cgtggaaaca
      541 tctgctaaaa cacgagctaa tgttgacaag gtattttttg atttaatgag agaaattcga
      601 gcgagaaaga tggaagacag caaagaaaag aatggaaaaa agaagaggaa aagtttagcc
      661 aagagaatca gagaaagatg ctgcatttta taagatccgg ctgctaacaa agcccgaaag
      721 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
      781 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga
      841 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
      901 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
      961 actgtgataa actaccgcat taaagctagc ttatcgatga taagctgtca aacatgagaa
     1021 ttaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1081 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1141 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1201 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1261 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1321 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1381 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1441 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     1501 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1561 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1621 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1681 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1741 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     1801 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1861 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1921 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     1981 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2041 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2101 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2161 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2221 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2281 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2341 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2401 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2461 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2521 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2581 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2641 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2701 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2761 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2821 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2881 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2941 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3001 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3061 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     3121 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3181 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3241 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3301 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3361 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3421 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3481 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3541 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     3601 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3661 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3721 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3781 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     3841 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     3901 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     3961 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     4021 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     4081 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     4141 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     4201 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag
     4261 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     4321 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     4381 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     4441 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     4501 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     4561 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     4621 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     4681 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     4741 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     4801 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4861 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     4921 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     4981 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     5041 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     5101 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     5161 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     5221 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     5281 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     5341 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     5401 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     5461 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     5521 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     5581 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     5641 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     5701 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     5761 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     5821 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     5881 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     5941 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     6001 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     6061 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     6121 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     6181 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     6241 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     6301 gaaataattt tgtttaaact ttaagaagga gatatacata tg
//
