LOCUS       sequence-140408-        7105 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template to generate Cdk2-T160A-siR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7105)
  AUTHORS   Kaulich M, Lee YJ, Lonn P, Springer AD, Meade BR, Dowdy SF
  TITLE     Efficient CRISPR-rAAV engineering of endogenous genes to study 
            protein function by allele-specific RNAi.
  JOURNAL   Nucleic Acids Res. 2015 Apr 20;43(7):e45. doi: 10.1093/nar/gku1403. 
            Epub 2015 Jan 13.
  PUBMED    25586224
REFERENCE   2  (bases 1 to 7105)
  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: "Nucleic 
            Acids Res."; date: "2015-04-20"; volume: "43(7):e45. doi"; pages: " 
            10.1093/nar/gku1403. Epub 2015 Jan 13"
COMMENT     Sequence Label: pAAV-SEPT_Cdk2-T160A-siR
FEATURES             Location/Qualifiers
     source          1..7105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     primer_bind     1245..1261
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1245..1261
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(1263..1296)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     intron          1334..1563
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1546..1565
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    1692..2154
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1748..1765)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1975..1994
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2168..2962
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2222..2241)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2832..2851
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2984..3118
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3008..3027)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3062..3081
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(3215..3248)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   4368..4508
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4368..4497
                     /label=AAV2 ITR
     rep_origin      4583..5038
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4670..4689)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4880..4901
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5055..5074)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5174..5196
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5234..5252)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5320..5424
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5425..5493,5494..6285)
                     /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(5643..5662)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6456..7044
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6945..6964
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca ccggtggcgc gccggagagg tgggttgggg
      181 gccagtagaa ggtgaagagt atacttatac tccctgggga gagtataggg tggtgtggaa
      241 tccatggaaa actttcttcc caaactgagc cggatcgtgc ccccaaatgt gcgactacag
      301 actcggggag agaaaggagg tctctgagat gaggtccaag actctccatg gagtggagtt
      361 atgtgggaac cggcgagaat cgcctttctg aatgaagagc cctcttcact gccccaccct
      421 caccttagaa ttctctcctc tttccaaaga atggcagttg aacctcactg gcccctctgg
      481 ggaggctggg ggctactcct gcattttttc ccctccatta cagtctccct gcttcacctt
      541 caccaggcgg ctttacttac ctacccctgg gaaaagagga gataatggcc ttaatatatc
      601 caaaaaccac accccgacta cccaagaatt agctcttacc atcacccttt ctcttctctc
      661 actttcctag ggggcgctgg gtggtgtctc ctcgggagaa agaaatgact aggtgggggg
      721 gaaaggaata tttgtaacca tattcccatc tctgctttcc caacctctcc aagtgagact
      781 gagggtgtgc ccagtactgc catccgagag atctctctgc ttaaggaatt gaatcacccg
      841 aacattgtca agtaagtatg cgtctgagag gtgatccagc tggaaaggag gataagttct
      901 gtctgtacag tgtgggcatt tctctctctc acacacctcc atttcctcaa actttccttc
      961 tctaggctgc tggatgtcat tcacacagaa aataaactct acctggtttt tgaatttctg
     1021 caccaagatc tcaagaaatt catggatgcc tctgctctca ctggcattcc tcttcccctc
     1081 atcaaggtaa tgcttctcat cagctcctct catcatgggc atgtcttggg ggactggtgg
     1141 caggcaattc agggtgatat tttatgattt tggcctcctt ctgagccctc atctcctata
     1201 cacacacact ccccttcttt ttgtgtctcc ttccctgcga gctctctaga actagtggat
     1261 ccataacttc gtatagcata cattatacga agttatggaa ttaattcgct gtctgcgagg
     1321 gccagctgtt ggggtgagta ctccctctca aaagcgggca tgacttctgc gctaagattg
     1381 tcagtttcca aaaacgagga ggatttgata ttcacctggc ccgcggtgat gcctttgagg
     1441 gtggccgcgt ccatctggtc agaaaagaca atctttttgt tgtcaagctt gaggtgtggc
     1501 aggcttgaga tctggccata cacttgagtg acaatgacat ccactttgcc tttctctcca
     1561 caggtgtcca ctcccaggtc caactgcagg tcgagcgtcg agaacgttac tggccgaagc
     1621 cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct
     1681 tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt
     1741 ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct
     1801 ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc
     1861 ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag
     1921 gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc
     1981 tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga
     2041 tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt
     2101 ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataagcttgc
     2161 cacaaccatg gctgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     2221 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     2281 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     2341 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     2401 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     2461 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     2521 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     2581 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     2641 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     2701 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     2761 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     2821 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     2881 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     2941 ccttcttgac gagttcttct gagcgggact ctggggttcg ggggatccag acatgataag
     3001 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     3061 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     3121 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggttttttc
     3181 tagggggccc cggcgaggcc gggatgagtt gggaataact tcgtatagca tacattatac
     3241 gaagttatcc aaattttaag gtaccactgt gcatatgtat gaattcgata tcaagcttat
     3301 cgatccctag ggttggactg aacaatcaaa gttgaaactc tagtgagtca acctagcaac
     3361 tcaggtggga ggtcagatga aactcagata aacgggattt gagagcactt ggtaaattcc
     3421 tccaaaaagc ccttccattt ggtggaagac ctagctagtg agtccctatt gtctatttta
     3481 gggctggatt cttcactccc agagctactt tcaatctatt aacaaacatt ttttcaatgc
     3541 acaggatgta gaaaagggat ggaaaattga gtaagacttg gtccttatcc tctctgggct
     3601 gacagtccat tgggagaaat agcttgtaaa tatgtaacta taatccaaca taataaaggc
     3661 tttagtagag ttttaggggc acagagcaaa cccagtctgc tcactgtaat ggagaaacac
     3721 agtcctctct ttctcctttg tcagagctat ctgttccagc tgctccaggg cctagctttc
     3781 tgccattctc atcgggtcct ccaccgagac cttaaacctc agaatctgct tattaacaca
     3841 gagggggcca tcaagctagc agactttgga ctagccagag cttttggagt tcctgttcgt
     3901 acttacgccc atgaggtgag tccctttatg tcttttttct ctgagcttcc caagaggtgt
     3961 taactagggt attcacaaag ttactaaaaa tatctggcta acagtttctt actaggtaga
     4021 aataatctct tgacatccta aagagtctta gggtatgcat ggaattcata ctgtgttgct
     4081 aactgggccc acacctgtaa taccaatact ttgggaggct gaggtgggag gatcacttga
     4141 gcccaggagt tcgagaccat catgggcaac atagcgagac cccatctcta caaatctaca
     4201 aaaagaaaaa atttagaaat aaaattatga ccaatttgtc tcaagttttt ccaggaagat
     4261 ctcaaattag gggttcagtc cagaactatg gactggaaat cagtgggagg ggaaagatga
     4321 tggagggaag gaaacgtcga cctcgagggg ccccggccgg cggccgcagg aacccctagt
     4381 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     4441 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     4501 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4561 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4621 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     4681 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4741 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     4801 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4861 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4921 tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4981 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     5041 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     5101 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5161 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5221 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5281 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5341 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5401 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5461 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5521 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5581 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5641 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5701 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5761 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5821 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5881 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5941 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6001 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6061 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6121 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6181 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6241 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6301 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6361 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6421 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6481 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6541 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6601 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6661 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6721 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6781 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6841 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6901 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6961 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7021 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7081 ttttgctggc cttttgctca catgt
//
