LOCUS       sequence-501656-        5017 bp    DNA     circular SYN 03-MAR-2026
DEFINITION  all-in-one HDRT-based knockin-knockout (KIKO) design to insert 
            mCherry (=knockin) at gThy1 cut site, disrupting Thy1 expression 
            (=knockout).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5017)
  TITLE     Krummel Lab plasmids
REFERENCE   2  (bases 1 to 5017)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 3, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pscAAV-KIKO-U6-gThy1-p2a-mCherry-STOP-pA-400
FEATURES             Location/Qualifiers
     source          1..5017
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      3..591
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     492..511
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    879..900
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(915..920,921..938,939..945)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    953..969
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     958..980
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     977..993
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     977..993
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1189..1437
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1189..1209
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1360..1379
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1459..1534
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1956..2012
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2013..2723
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2158..2176)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2426..2445
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2744..2763
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3394..3411)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3394..3410)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3403..3425)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3619..3638)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3738..3760
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3798..3816)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3884..3988
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3989..4057,4058..4849)
                     /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(4207..4226)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
       61 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
      121 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
      181 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
      241 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
      301 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
      361 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
      421 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      481 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      541 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      601 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      661 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      721 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      781 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      841 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      901 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      961 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct ctcgagatct
     1021 agaaagcttc ccggggggat ctgggccact ccctctctgc gcgctcgctc gctcactgag
     1081 gccgggcgac caaaggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg
     1141 ccaactccat cactaggggt tcctggaggg gtggagtcgt gacctaggga gggcctattt
     1201 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt
     1261 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     1321 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     1381 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggc
     1441 tggtcacctt ctgccctcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     1501 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ggtacctgtg gtacacacct
     1561 ttaatctcag cactcaggag gcagagtcag gtggatctgt tagtctgaag ccagcctggt
     1621 ctacagagca aattccagga cagccagagc tattctcaag atagagaatc cctttcttga
     1681 aaaaaccatt taaaaacaaa aacaaaagca acacactcct cttgatctcc tgttcttgaa
     1741 acacattgtt gggacccaga acttcagtag attgatggaa gttggagtct gcaagtggtg
     1801 gaacatccca ccaatacctc aagggcgagt gcaaacccca catcccccca gctcaagtct
     1861 acttttcctg caggtgggag gccccgggtc tgtgtctccc caaattcaga gaaggcactg
     1921 ctgtgccagt cttgcaggtg tcccgaggca gcggcgccac caacttcagc ctgctgaagc
     1981 aggccggcga cgtggaggag aaccccggcc ccatggtgag caagggcgag gaggataaca
     2041 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     2101 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     2161 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     2221 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     2281 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     2341 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     2401 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     2461 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     2521 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     2581 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     2641 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     2701 gcatggacga gctgtacaag taagcggccg ctgctttatt tgtgaaattt gtgatgctat
     2761 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     2821 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagggcag aaggtgacca
     2881 gcctgacagc ctgcctggtg aaccaaaacc ttcgcctgga ctgccgccat gagaataaca
     2941 ccaaggataa ctccatccag catgagttca gcctgacccg agagaagagg aagcacgtgc
     3001 tctcaggcac ccttgggata cccgagcaca cgtaccgctc ccgcgtcacc ctctccaacc
     3061 agccctatat caaggtcctt accctagcca acttcaccac caaggatgag ggcgactact
     3121 tttgtgagct tcaagtctcg ggcgcgaatc ccatgagctc caataaaagt atcagtgtgt
     3181 atagaggtga gactggttcc cagaaagata aaatgtccag gttagccagg ctggggtagc
     3241 caatactagt ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     3301 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagagag
     3361 ggacagatcc gggcccgcat gcgtcgacaa ttcactggcc gtcgttttac aacgtcgtga
     3421 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     3481 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     3541 tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3601 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     3661 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3721 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3781 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     3841 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3901 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3961 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4021 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4081 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4141 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4201 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4261 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4321 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4381 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4441 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4501 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     4561 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4621 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4681 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     4741 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4801 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4861 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4921 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4981 ctgagcgtca gaccccgtag aaaagatcaa aggatct
//
