LOCUS       sequence-337906-        5509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription for rescue experiments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5509)
  AUTHORS   Lee YR, Kim SH, Ben-Mahmoud A, Kim OH, Choi TI, Lee KH, Ku B, Eum J,
            Kee Y, Lee S, Cha J, Won D, Lee ST, Choi JR, Lee JS, Kim HD, Kim HG,
            Bonkowsky JL, Kang HC, Kim CH
  TITLE     Eif2b3 mutants recapitulate phenotypes of vanishing white matter 
            disease and validate novel disease alleles in zebrafish.
  JOURNAL   Hum Mol Genet. 2021 Apr 27;30(5):331-342. doi: 10.1093/hmg/ddab033.
  PUBMED    33517449
REFERENCE   2  (bases 1 to 5509)
  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"; journalName: 
            "Hum Mol Genet"; date: "2021-04-27- 27"; volume: "30"; issue: "5"; 
            pages: "331-342"
COMMENT     Sequence Label: EIF2B3-S369fs
FEATURES             Location/Qualifiers
     source          1..5509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1355..1366
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     polyA_signal    2539..2673
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2563..2582)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2617..2636
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2786..2806)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2786..2804)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2825..2841)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2825..2841)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2838..2860)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2849..2865
                     /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)."
     promoter        complement(join(2873..2879,2880..2897,2898..2903))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2918..2939
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3056..3073)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3227..3815)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3307..3326)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3986..4777,4778..4846))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4609..4628
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4847..4951)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4977..5432)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5114..5135)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5326..5345
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     1021 gacactatag aatacaagct acttgttctt tttgcaggat cccatcgatg gccgcacaca
     1081 agctggctga cccggtttgt aaaaatggaa tttcaagcag tagtgatggc agtaggtgga
     1141 ggatctcgga tgacagacct aacttccagc attcccaaac ctctgcttcc agttgggaac
     1201 aaacctttaa tttggtaccc attgaacctg cttgagcgtg ttggatttga agaagtcatt
     1261 gtggttacaa ccagggatgt tcaaaaggct ctatgtgcag aattcaagat gaaaatgaag
     1321 ccagatattg tgtgtattcc tgatgatgct gacatgggaa ctgcagattc tttgcgctac
     1381 atatatccaa aacttaagac agatgtgctg gtgctgagct gtgatctgat aacagacgtt
     1441 gccttacatg aggttgtgga cctgtttaga gcttatgatg catcacttgc tatgttgatg
     1501 agaaaaggcc aagatagcat agaacctgtt cccggtcaaa aggggaaaaa aaaagcagtg
     1561 gagcagcgtg acttcattgg agtggacagc acaggaaaga ggctgctctt catggctaat
     1621 gaagcagact tggatgaaga gctggtcatt aagggatcca tcctacagaa gcatcctaga
     1681 atacgtttcc acacgggtct tgtggatgcc cacctctact gtttgaaaaa atacatcgtg
     1741 gacttcctaa tggaaaatgg gtcaataact tctatccgga gtgaactgat tccatattta
     1801 gtgagaaaac agttttcctc agcttcctca caacagggac aagaagaaaa agaggaggat
     1861 ctaaagaaaa aggagctgaa gtccttagat atctacagtt ttataaaaga agccaataca
     1921 ctgaacctgg ctccctatga tgcctgctgg aatgcctgtc gaggagacag gtgggaagac
     1981 ttgtccagat cacaggtgcg ctgctatgtc cacatcatga aagaggggct ctgctctcga
     2041 gtgagcacac tgggactcta catggaagca aacagacagg tgcccaaatt gctgtctgct
     2101 ctctgtccag aagaaccacc agtccattcg tcagcccaga ttgtcagcaa acacctggtt
     2161 ggagttgaca gcctcattgg gccagagaca cagattggag agaagtcatc gctcagtcat
     2221 tggctcatcc tgtctcataa aagatagagt gactattacc aattgccttc tcatgaactc
     2281 agtcactgtg gaggaaggaa gcaatatcca aggcagtgtc atctgcaaca atgctgtgat
     2341 cgagaagggt gcagacatca aggactgctt gattggaagt ggccagagga ttgaagccaa
     2401 agctaaacga gtgaatgagg tgatcgtggg gaatgaccag ctcatggaga tctgagttct
     2461 gagcaagtca gactccttcc ttttggcctc caaagccaca gatgttggct ctagaactat
     2521 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     2581 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     2641 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     2701 tttcaggttc agggggaggt gtgggaggtt ttttaattcg cggccgcggc gccaatgcat
     2761 tgggcccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     2821 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     2881 gagccgggag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     2941 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3001 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3061 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3121 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3181 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3241 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3301 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3361 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3421 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3481 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3541 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3601 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3661 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3721 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3781 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3841 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3901 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3961 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4021 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4081 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4141 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4201 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4261 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4321 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4381 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4441 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4501 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4561 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4621 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4681 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4741 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4801 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4861 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4921 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     4981 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5041 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5101 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5161 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5221 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     5281 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     5341 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5401 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg
     5461 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagtcgacc
//
