LOCUS       sequence-478213-        4791 bp    DNA     circular SYN 11-JUL-2025
DEFINITION  KI:Lmnb1 Donor:3xV5 KO:Template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4791)
  TITLE     AAV backbone plasmids for knock-in or knockout CRISPR genome editing
REFERENCE   2  (bases 1 to 4791)
  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;Lmnb1 Donor;3xV5 KO;Template
FEATURES             Location/Qualifiers
     source          1..4791
                     /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(1851..1892)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1902..1943)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1953..1994)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     repeat_region   2054..2194
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2054..2183
                     /label=AAV2 ITR
     rep_origin      2269..2724
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2356..2375)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2566..2587
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2741..2760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2860..2882
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2920..2938)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3006..3110
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3111..3179,3180..3971)
                     /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(3329..3348)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4142..4730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4631..4650
                     /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 ctgtcttcat catggccgac 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 cttgttggtt
      901 cgcattatcc gaaccatgtt 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 gaaacatgta agcgtcgcaa
     1321 gaagacgggt 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 accggacgtg
     1681 acaagttcac ataagtttaa gagctaagct cagaagagca tagcaagttt aaataaggct
     1741 agtccgtact caacttgaaa aagtggcagc cgagtcggct gctttttttg gtctgagacg
     1801 ccgcgccatt atgtgaactt gtcacgtcgg atccctagct agctagctag cgtggagtcc
     1861 aggcccaaca gagggttagg gataggctta ccttgttgct gtgtgctatc cagaccgaga
     1921 agtgggtttg gaatgggctt accctgctgc tgagtactat caagtccaag gagggggttc
     1981 ggtataggtt ttccttctcg agccattatg tgaacttgtc acgtccacgc gtgtgtctag
     2041 actgcgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     2101 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     2161 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     2221 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     2281 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2341 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2401 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2461 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     2521 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2581 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     2641 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2701 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     2761 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2821 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2881 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     2941 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     3001 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3061 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3121 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3181 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3241 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3301 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3361 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3421 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3481 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3541 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3601 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3661 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3721 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3781 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3841 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3901 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3961 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4021 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4081 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4141 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4201 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4261 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4321 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4381 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4441 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4501 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4561 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4621 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4681 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4741 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
