LOCUS       sequence-336112-        5594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor shuttle plasmid to enable ligation of a second U6-GuideRNA 
            casssette into any pX458 family backbone via digestion of both 
            plasmids with XbaI and KpnI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5594)
  AUTHORS   Kaufman ML, Goodson NB, Park KU, Schwanke M, Office E, Schneider SR,
            Abraham J, Hensley A, Jones KL, Brzezinski JA
  TITLE     Initiation of Otx2 expression in the developing mouse retina 
            requires a unique enhancer and either Ascl1 or Neurog2 activity.
  JOURNAL   Development. 2021 Jun 15;148(12). pii: 269198. doi: 
            10.1242/dev.199399. Epub 2021 Jun 18.
  PUBMED    34143204
REFERENCE   2  (bases 1 to 5594)
  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: 
            "Development."; date: "2021-06-15"; volume: "148(12). pii: 269198. 
            doi"; pages: " 10.1242/dev.199399. Epub 2021 Jun 18"
COMMENT     Sequence Label: pX458-Dual-Guide-Donor-ShuttleOnly
FEATURES             Location/Qualifiers
     source          1..5594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      168..623
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(255..274)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     465..486
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(640..659)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     759..781
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(819..837)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        905..1009
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1010..1078,1079..1870)
                     /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     complement(1228..1247)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2041..2629
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2530..2549
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2697..2945
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2868..2887
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2964..3039
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3287..4469
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3518..4460
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4417..4437
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4475..4484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4484..4549
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4556..4576
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     repeat_region   join(5547..5594,1..93)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(5547..5594,1..82)
                     /label=AAV2 ITR
ORIGIN
        1 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
       61 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
      121 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
      181 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
      241 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
      301 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
      361 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
      421 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
      481 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
      541 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
      601 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
      661 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
      721 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
      781 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
      841 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
      901 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
      961 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1021 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1081 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1141 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1201 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     1261 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1321 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1381 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     1441 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     1501 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     1561 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     1621 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     1681 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     1741 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     1801 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     1861 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     1921 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     1981 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2041 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2101 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2161 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2221 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2281 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2341 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2401 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2461 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2521 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2581 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2641 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcatctag acatgtgagg
     2701 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     2761 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     2821 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     2881 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     2941 acaccgggtc ttcgagaaga cctgttttag agctagaaat agcaagttaa aataaggcta
     3001 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa
     3061 atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat
     3121 ggctctacag gtaccagtaa ttcatacaaa aggactcgcc cctgccttgg ggaatcccag
     3181 ggaccgtcgt taaactccca ctaacgtaga acccagagat cgctgcgttc ccgccccctc
     3241 acccgcccgc tctcgtcatc actgaggtgg agaagagcat gcgtgaggct ccggtgcccg
     3301 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     3361 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     3421 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     3481 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
     3541 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacgcccct
     3601 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
     3661 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cttgggcgct
     3721 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
     3781 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
     3841 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
     3901 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
     3961 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
     4021 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     4081 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     4141 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     4201 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     4261 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     4321 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     4381 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     4441 gtttttttct tccatttcag gtgtcgtgac cggtgccacc atggactata aggaccacga
     4501 cggagactac aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa
     4561 gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg
     4621 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     4681 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     4741 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     4801 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     4861 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     4921 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     4981 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     5041 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     5101 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     5161 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     5221 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     5281 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     5341 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     5401 ggacacctac gacgacgacc tggacaacct gctggccttt tgctcatcta gacatgggaa
     5461 ataggccctc acatgtctag atgagcaaaa ggccagcaag ggggaggatt gggaagagaa
     5521 tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc
     5581 tctgcgcgct cgct
//
