LOCUS       sequence-140404-        7077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template to generate Cdk2-T160E-siR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7077)
  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 7077)
  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-T160E-siR
FEATURES             Location/Qualifiers
     source          1..7077
                     /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   4340..4480
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4340..4469
                     /label=AAV2 ITR
     rep_origin      4555..5010
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4642..4661)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4852..4873
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5027..5046)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5146..5168
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5206..5224)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5292..5396
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5397..5465,5466..6257)
                     /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(5615..5634)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6428..7016
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6917..6936
                     /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 gcatatgccc tagggttgga ctgaacatca
     3301 aagttgaaac tctagtgagt caacctagca actcaggtgg gaggtcagat gaaactcaga
     3361 taaacgggat ttgagagcac ttggtaaatt cctccaaaaa gcccttccat ttggtggaag
     3421 acctagctag tgagtcccta ttgtctattt tagggctgga ttcttcactc ccagagctac
     3481 tttcaatcta ttaacaaaca ttttttcaat gcacaggatg tagaaaaggg atggaaaatt
     3541 gagtaagact tggtccttat cctctctggg ctgacagtcc attgggagaa atagcttgta
     3601 aatatgtaac tataatccaa cataataaag gctttagtag agttttaggg gcacagagca
     3661 aacccagtct gctcactgta atggagaaac acagtcctct ctttctcctt tgtcagagct
     3721 atctgttcca gctgctccag ggcctagctt tctgccattc tcatcgggtc ctccaccgag
     3781 accttaaacc tcagaatctg cttattaaca cagagggggc catcaagcta gcagactttg
     3841 gactagccag agcttttgga gtccctgttc gtacttacga gcatgaggtg agtcccttta
     3901 tgtctttttt ctctgagctt cccaagaggt gttaactagg gtattcacaa agttactaaa
     3961 aatatctggc taacagtttc ttactaggta gaaataatct cttgacatcc taaagagtct
     4021 tagggtatgc atggaattca tactgtgttg ctaactgggc ccacacctgt aataccaata
     4081 ctttgggagg ctgaggtggg aggatcactt gagcccagga gttcgagacc atcatgggca
     4141 acatagcgag accccatctc tacaaatcta caaaaagaaa aaatttagaa ataaaattat
     4201 gaccaatttg tctcaagttt ttccaggaag atctcaaatt aggggttcag tccagaacta
     4261 tggactggaa atcagtggga ggggaaagat gatggaggga aggaaacgtc gacctcgagg
     4321 ggccccggcc ggcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     4381 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     4441 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     4501 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     4561 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4621 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4681 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4741 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     4801 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4861 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga
     4921 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4981 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     5041 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5101 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5161 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     5221 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     5281 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5341 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5401 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5461 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5521 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5581 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5641 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5701 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5761 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5821 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5881 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5941 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6001 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6061 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6121 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6181 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6241 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6301 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6361 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6421 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6481 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6541 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     6601 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6661 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6721 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6781 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6841 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6901 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6961 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7021 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
