LOCUS       sequence-478525-        4778 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  KI:Sptbn4 Donor:3xV5 KO:Template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4778)
  TITLE     AAV backbone plasmids for knock-in or knockout CRISPR genome editing
REFERENCE   2  (bases 1 to 4778)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pHnS KI;Sptbn4 Donor;3xV5 KO;Template
FEATURES             Location/Qualifiers
     source          1..4778
                     /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             1836..1877
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1887..1928
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1938..1979
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     repeat_region   2041..2181
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2041..2170
                     /label=AAV2 ITR
     rep_origin      2256..2711
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2343..2362)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2553..2574
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2728..2747)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2847..2869
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2907..2925)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2993..3097
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3098..3166,3167..3958)
                     /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(3316..3335)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4129..4717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4618..4637
                     /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 accgagcgta
     1681 gggccagcgg gcgcgtttaa gagctaagct cagaagagca tagcaagttt aaataaggct
     1741 agtccgtact caacttgaaa aagtggcagc cgagtcggct gctttttttg gtctgagacg
     1801 ccgcgccggc gcccgctggc cctacgctct cgagaggaaa acctataccg aaccccctcc
     1861 ttggacttga tagtactcag cagcagggta agcccattcc aaacccactt ctcggtctgg
     1921 atagcacaca gcaacaaggt aagcctatcc ctaaccctct gttgggcctg gactccacgt
     1981 aggggatccc cggcgcccgc tggccctacg ctcacgcgtg tgtctagact gcgcggccgc
     2041 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     2101 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     2161 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     2221 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     2281 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     2341 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2401 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2461 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2521 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2581 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     2641 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2701 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2761 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2821 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2881 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2941 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     3001 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3061 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3121 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3181 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3241 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3301 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3361 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3421 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3481 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3541 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3601 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3661 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3721 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3781 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3841 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3901 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3961 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4021 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4081 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4141 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4201 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4261 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4321 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4381 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4441 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4501 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4561 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4621 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4681 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4741 acggttcctg gccttttgct ggccttttgc tcacatgt
//
