LOCUS       sequence-478297-        4801 bp    DNA     circular SYN 11-JUL-2025
DEFINITION  KI:Actb Donor:3xV5 KO:Dlg4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4801)
  TITLE     AAV backbone plasmids for knock-in or knockout CRISPR genome editing
REFERENCE   2  (bases 1 to 4801)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pHnS KI;Actb Donor;3xV5 KO;Dlg4
FEATURES             Location/Qualifiers
     source          1..4801
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(778..811)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     815..835
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     1060..1080
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1231..1250
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        1455..1669
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     1590..1609
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     CDS             complement(1837..1878)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1888..1929)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1939..1980)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     regulatory      1980..1989
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     repeat_region   2064..2204
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2064..2193
                     /label=AAV2 ITR
     rep_origin      2279..2734
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2366..2385)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2576..2597
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2751..2770)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2870..2892
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2930..2948)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3016..3120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3121..3189,3190..3981)
                     /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(3339..3358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4152..4740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4641..4660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgccg gtaggtccct cgacgttaac cgtgaccgag cttgtctgcc
      181 cgcgcagtcc actagacaga cgcgcagagg tcgggcgggc ctagggccaa tttcccatgg
      241 ttcccttcat ttgcatatat gatgtaataa ggttatggag actattaaac ttagccctaa
      301 tcaaactata tgatgataat gtgtgtggta caaaaggtca taacttatta tatactttga
      361 aacttaaaaa agggttacag tttagtcacc ataactgtaa aattttttct attcttagct
      421 ttatatagtt cttgagaggc catgtttatg aggcggatcg tgatccatcg gtttaagagc
      481 taagctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt
      541 ggcaccgagt cggtgctttt tttccagtct gactatcacg atggatccga cgcgccatct
      601 ctaggcccgc gccggccccc tcgcacggac ttgtgggaga agctcggcta ctcccctgcc
      661 ccggttaatt tgcatataat atttcctagt aactatagag gcttaatgtg cgataaaaga
      721 cagataatct gttcttttta atactagcta cattttacat gataggcttg gatttctata
      781 acttcgtata gcatacatta tacgaagtta taaacagcac aaaaggaaac tcaccctaac
      841 tgtaaagtaa ttgtgtgttt tgagactata agtatccctt ggagaaccac cttgttgccg
      901 caatgccagt cacgaacgtt tgagagctaa gcagaaagct gcatagcaag ttcaaataag
      961 gctagtccgt acacaacttg aaaaagtggc agccgagtcg gctgcttttt ttgctcgaat
     1021 ctacactcag ctatggcgcg ccccaaggtc gggcaggaag agggcctatt tcccatgatt
     1081 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
     1141 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     1201 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     1261 tatttcgatt tcttggcttt atatatcttg tggaaagcca gaaacatgcg tcatgcacga
     1321 ggatgccggt ttcagagcta agcacaagag tgcatagcaa gttgaaataa ggctagtccg
     1381 tttacaactt gaaaaagtgg cacccgagtc gggtgctttt tttctcgagt actaggatcc
     1441 attaggcggt ctaggaacgc tgacgtcatc aacccgctcc aaggaatcgc gggcccagtg
     1501 tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag gaagatggct gtgagggaca
     1561 ggggagtggc gccctgcaat atttgcatgt cgctatgtgt tctgggaaat caccataaac
     1621 gtgaaatgtc tttggatttg ggaatcttat aagttctgta tgagaccacc accgcgcagc
     1681 gatatcgtca tccagtttaa gagctaagct cagaagagca tagcaagttt aaataaggct
     1741 agtccgtact caacttgaaa aagtggcagc cgagtcggct gctttttttg gtctgagacg
     1801 ccgcgccatg gatgacgata tcgctgcggt gatccccgtg gagtccaggc ccaacagagg
     1861 gttagggata ggcttacctt gttgctgtgt gctatccaga ccgagaagtg ggtttggaat
     1921 gggcttaccc tgctgctgag tactatcaag tccaaggagg gggttcggta taggttttcc
     1981 catggtggcc tcgagtagct agctagctag ccccatggat gacgatatcg ctgcgcacgc
     2041 gtgtgtctag actgcgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     2101 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     2161 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     2221 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     2281 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2341 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2401 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2461 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     2521 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2581 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     2641 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2701 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     2761 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     2821 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     2881 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     2941 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     3001 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3061 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3121 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3181 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3241 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3301 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3361 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3421 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3481 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3541 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3601 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3661 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3721 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3781 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3841 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3901 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3961 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4021 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4081 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4141 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4201 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4261 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4321 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4381 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4441 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4501 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4561 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4621 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4681 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4741 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4801 t
//
