LOCUS       sequence-238091-        6341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of RALA WT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6341)
  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 6341)
  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 WT
FEATURES             Location/Qualifiers
     source          1..6341
                     /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(742..760)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      756..803
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1041..1059)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1127..1231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1232..1300,1301..2092)
                     /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(1450..1469)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2263..2851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2752..2771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3005..3022
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3037..3176
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3102..3121)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3262..3284
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3278..3469)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4781..5863)
                     /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     5825..5844
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5864..5941)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6147..6166
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6250..6269
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6250..6268
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6269..6293
                     /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 ggttccgggc ttgtaccgcg cggatctcat
       61 atgggatccc acatggctgc aaataagccc aagggtcaga attctttggc tttacacaaa
      121 gtcatcatgg tgggcagtgg tggcgtgggc 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 gagaagatgc tgcattttat aagatccggc tgctaacaaa gcccgaaagg
      721 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
      781 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag
      841 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
      901 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
      961 ctgtgataaa ctaccgcatt aaagctagct tatcgatgat aagctgtcaa acatgagaat
     1021 taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     1081 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1141 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     1201 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     1261 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     1321 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1381 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     1441 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     1501 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     1561 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     1621 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     1681 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1741 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     1801 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1861 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1921 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     1981 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     2041 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     2101 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     2161 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     2221 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2281 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2341 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2401 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2461 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2521 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2581 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2641 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2701 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2761 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2821 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2881 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     2941 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3001 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3061 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3121 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3181 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3241 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3301 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3361 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     3421 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3481 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3541 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     3601 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     3661 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     3721 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     3781 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     3841 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     3901 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     3961 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     4021 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     4081 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     4141 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     4201 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt
     4261 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     4321 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     4381 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     4441 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     4501 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     4561 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     4621 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     4681 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     4741 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4801 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4861 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     4921 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     4981 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5041 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     5101 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5161 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5221 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5281 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5341 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5401 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     5461 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     5521 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     5581 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     5641 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     5701 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     5761 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     5821 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     5881 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     5941 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     6001 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     6061 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     6121 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     6181 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     6241 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     6301 aaataatttt gtttaaactt taagaaggag atatacatat g
//
