LOCUS       sequence-337908-        5515 bp    DNA     circular SYN 15-APR-2025
DEFINITION  In vitro transcription for rescue experiments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5515)
  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 5515)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-C424G
FEATURES             Location/Qualifiers
     source          1..5515
                     /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    2545..2679
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2569..2588)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2623..2642
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2792..2812)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2792..2810)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2831..2847)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2831..2847)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2844..2866)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2855..2871
                     /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(2879..2885,2886..2903,2904..2909))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2924..2945
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3062..3079)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3233..3821)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3313..3332)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3992..4783,4784..4852))
                     /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     4615..4634
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4853..4957)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4983..5438)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5120..5141)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5332..5351
                     /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 cattaagcgc
     2221 tcagtcattg gctcatcctg tctcataaaa gatagagtga ctattaccaa ttgccttctc
     2281 atgaactcag tcactggagg aaggaagcaa tatccaaggc agtgtcatct gcaacaatgc
     2341 tgtgatcgag aagggtgcag acatcaagga ctgcttgatt ggaagtggcc agaggattga
     2401 agccaaagct aaacgagtga atgaggtgat cgtggggaat gaccagctca tggagatctg
     2461 agttctgagc aagtcagact ccttcctttt ggcctccaaa gccacagatg ttggctctag
     2521 aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga tgagtttgga
     2581 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     2641 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     2701 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcgcggc cgcggcgcca
     2761 atgcattggg cccggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     2821 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     2881 acatacgagc cgggagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     2941 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3001 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3061 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3121 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3181 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3241 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3301 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3361 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3421 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3481 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3541 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3601 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3661 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3721 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3781 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3841 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3901 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3961 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4021 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4081 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4141 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4201 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4261 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4321 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4381 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4441 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4501 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4561 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4621 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4681 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4741 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4801 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4861 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4921 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4981 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     5041 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5101 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5161 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5221 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5281 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5341 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5401 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
     5461 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagt cgacc
//
