LOCUS       sequence-238094-        6342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of RALA G23D.
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 G23D
FEATURES             Location/Qualifiers
     source          1..6342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             74..91
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             113..130
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(816..834)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      830..877
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1115..1133)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1201..1305
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1306..1374,1375..2166)
                     /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(1524..1543)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2337..2925
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2826..2845
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3079..3096
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3111..3250
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3176..3195)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3336..3358
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3352..3543)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4855..5937)
                     /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     5899..5918
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5938..6015)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6221..6240
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6324..6342,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6324..6342
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaaac tttaagaagg
       61 agatatacat atgcatcatc atcatcatca cgtgaattcg ctcggttccg ggcttgtacc
      121 gcgcggatct catatgggat cccacatggc tgcaaataag cccaagggtc agaattcttt
      181 ggctttacac aaagtcatca tggtgggcag tgatggcgtg ggcaagtcag ctctgactct
      241 acagttcatg tacgatgagt ttgtggagga ctatgagcct accaaagcag acagctatcg
      301 gaagaaggta gtgctagatg gggaggaagt ccagatcgat atcttagata cagctgggca
      361 ggaggactac gctgcaatta gagacaacta cttccgaagt ggggaggggt tcctctgtgt
      421 tttctctatt acagaaatgg aatcctttgc agctacagct gacttcaggg agcagatttt
      481 aagagtaaaa gaagatgaga atgttccatt tctactggtt ggtaacaaat cagatttaga
      541 agataaaaga caggtttctg tagaagaggc aaaaaacaga gctgagcagt ggaatgttaa
      601 ctacgtggaa acatctgcta aaacacgagc taatgttgac aaggtatttt ttgatttaat
      661 gagagaaatt cgagcgagaa agatggaaga cagcaaagaa aagaatggaa aaaagaagag
      721 gaaaagttta gccaagagaa tcagagaaag atgctgcatt ttataagatc cggctgctaa
      781 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
      841 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
      901 atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg
      961 gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg
     1021 cgttagcaat ttaactgtga taaactaccg cattaaagct agcttatcga tgataagctg
     1081 tcaaacatga gaattaattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     1141 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     1201 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1261 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1321 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1381 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1441 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1501 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     1561 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1621 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     1681 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     1741 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     1801 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     1861 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     1921 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     1981 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2041 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2101 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2161 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2221 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2281 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2341 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2401 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2461 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2521 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2581 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2641 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2701 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2761 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2821 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2881 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2941 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3001 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3061 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3121 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3181 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3241 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3301 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3361 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     3421 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     3481 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     3541 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     3601 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     3661 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     3721 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     3781 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     3841 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     3901 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     3961 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc
     4021 aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt
     4081 ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct
     4141 tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg
     4201 ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gcctacaatc
     4261 catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt
     4321 ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg
     4381 tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa
     4441 gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg
     4501 tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     4561 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     4621 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     4681 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     4741 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     4801 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     4861 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4921 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     4981 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     5041 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     5101 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     5161 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     5221 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     5281 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     5341 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     5401 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     5461 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     5521 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     5581 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     5641 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     5701 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     5761 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     5821 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     5881 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     5941 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     6001 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     6061 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     6121 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     6181 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     6241 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     6301 tcgagatctc gatcccgcga aattaatacg actcactata gg
//
