LOCUS       sequence-478215-        5038 bp    DNA     circular SYN 11-JUL-2025
DEFINITION  KI:Sptbn1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5038)
  TITLE     AAV backbone plasmids for knock-in or knockout CRISPR genome editing
REFERENCE   2  (bases 1 to 5038)
  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: pAAV KI;Sptbn1 Donor;smFP-V5
FEATURES             Location/Qualifiers
     source          1..5038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        147..395
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     147..167
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     318..337
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(703..736)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     740..760
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     CDS             980..1021
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1031..1072
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1082..1123
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1655..1696
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1706..1747
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1766..1807
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1817..1858
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2072..2113
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2123..2164
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2174..2215
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     repeat_region   2301..2441
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2301..2430
                     /label=AAV2 ITR
     rep_origin      2516..2971
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2603..2622)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2813..2834
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2988..3007)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3107..3129
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3167..3185)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3253..3357
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3358..3426,3427..4218)
                     /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(3576..3595)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4389..4977
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4878..4897
                     /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 gcggccgctc acatgtgagg gcctatttcc catgattcct tcatatttgc
      181 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
      241 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
      301 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
      361 tggctttata tatcttgtgg aaaggacgaa acaccgcttc tccctcttgc caggacgttt
      421 aagagctaag ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
      481 aaaagtggca ccgagtcggt gcttttttga tccgacgcgc catctctagg cccgcgccgg
      541 ccccctcgca cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat
      601 ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct
      661 ttttaatact agctacattt tacatgatag gcttggattt ctataacttc gtatagcata
      721 cattatacga agttataaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg
      781 tgttttgaga ctataagtat cccttggaga accaccttgt tggaaacacc ggcgatcgta
      841 atcacccgag tgtttgagag ctaagcagaa agctgcatag caagttcaaa taaggctagt
      901 ccgtacacaa cttgaaaaag tggcagccga gtcggctgct tttttctgca ggcgatcgta
      961 atcacccgag tgggctcgag gaaaacctat accgaacccc ctccttggac ttgatagtac
     1021 tcagcagcag ggtaagccca ttccaaaccc acttctcggt ctggatagca cacagcaaca
     1081 aggtaagcct atccctaacc ctctgttggg cctggactcc acgggccaag gtgtgagtaa
     1141 gggcgaagaa ctgtttaccg gcgtggtccc aattctggtt gagctcgacg gtgacgttaa
     1201 cggacacaag tttagtgtgc gcggagaagg agagggagat gccaccaacg gaaaattgac
     1261 gctgaagttt atatgcacca cgggaaagct gccggtgccg tggccgacct tggttacaac
     1321 cctgggtggc ggagtccagt gtttcagccg ttacccagac cacatgaagc agcatgactt
     1381 ctttaagagt gctatgccgg agggatacgt gcaggaacgg acgatcagtt tcaaagatga
     1441 cggtacttac aagacgcgag ccgaggtgaa gttcgaggga gacaccttgg tgaacaggat
     1501 agaacttaag ggaattgatt tcaaggagga tggtaatatt ctgggtcaca agctggaata
     1561 caactttaac agccataacg tctacattac tgccgacaag cagaagaacg gcataaaggc
     1621 gaatttcaaa attcggcaca acgtggaagg tggtggcaag ccgatcccga atcccctgct
     1681 gggcttggat agcacgcagc aacagggaaa gcccatccct aaccccttgc tgggactcga
     1741 cagcaccgga ggccagcaag gcggtggcaa accgataccc aatcccctgc tcggcctgga
     1801 tagcacccag cagcaaggca agccaatccc caaccctctg ctgggcctcg actcgacagg
     1861 tggcgatgga agtgttcaac tggcggacca ctatcagcag aatacgccga tcggagatgg
     1921 ccccgtgctg ttgccggata atcactatct gtcgactcag agcgttctga gtaaggaccc
     1981 aaacgaaaaa cgagatcaca tggtgttgtt ggagttcgtg accgccgccg gcattacgtt
     2041 gggaatggac gagctgtaca agggccaagg aggcaagccc ataccaaatc ccttgctggg
     2101 cctcgacagc actcagcaac aaggaaagcc aataccaaat ccactgttgg gcctcgacag
     2161 tacgcaacag cagggcaagc ccatcccgaa cccgttgctg ggactggata gtacctaaac
     2221 cggtgctcga gaattcgata tcatagctag ctagctaggg atccgcgatc gtaatcaccc
     2281 gagtgggcta gaggggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     2341 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     2401 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     2461 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     2521 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2581 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2641 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2701 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     2761 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2821 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     2881 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2941 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     3001 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3061 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3121 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     3181 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     3241 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3301 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3361 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3421 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3481 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3541 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3601 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3661 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3721 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3781 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3841 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3901 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3961 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4021 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4081 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4141 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4201 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4261 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4321 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4381 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4441 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4501 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4561 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4621 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4681 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4741 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4801 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4861 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4921 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4981 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
