LOCUS       sequence-495771-        4605 bp    DNA     linear   UNA 18-DEC-2025
DEFINITION  Targeting vector for C-terminal knockin at ANXA1 locus to enable 
            reporting of expression by mScarlet and depletion of positive cells 
            with iCasp9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4605)
  TITLE     Stem cells actively suppress regenerative plasticity in human colon
REFERENCE   2  (bases 1 to 4605)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: TV_ANXA1-p2a-mScarlet-nls-p2a-icasp9_pgk-blast
FEATURES             Location/Qualifiers
     source          1..4605
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             488..544
                     /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             545..1237
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(675..693)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     943..962
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1244..1264
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1298..1354
                     /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             1355..1675
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             complement(2027..2038)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             2552..2578
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2611..2818
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2835..2868)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2886..3385
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2921..2944)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3318..3337
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3397..3444
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3463..3861
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3890..4024
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3914..3933)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3968..3987
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(4094..4127)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 tacgaaacta aacattccct tgaaggataa ctaaaatcta cccagaaaat gtgagaaaat
       61 tacaggcact atacatttct cttcttctgg tgagtgaatc aggtatctcc tcacataaga
      121 tatgagatta tattgtgtta tacttattta attttgtgtg atatttcatt catagaaaat
      181 aatggcacta atataaaaca taattaattc agaaaagttt gtaatctcag tcttgaaggc
      241 tattgctttt ttatgcaatg ataaaaaatc agttatctgt tgtatatatt gtttaattaa
      301 gtgaatggta atgtgtaatc tcatctacag ctaagtctaa aaataattct tctataagta
      361 aaaaaaaata gagaattatg gtttcgacta acattaagta tacctttttt tgaatcaaca
      421 ggatgaaacc aaaggagatt atgagaaaat cctggtggct ctttgtggag gaaacggcgg
      481 cagcggagcg acgaatttta gtctactgaa acaagcggga gacgtggagg aaaaccctgg
      541 acctgtgagc aagggcgagg cagtgatcaa ggagttcatg cggttcaagg tgcacatgga
      601 gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
      661 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tctcctggga
      721 catcctgtcc cctcagttca tgtacggctc cagggccttc atcaagcacc ccgccgacat
      781 ccccgactac tataagcagt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
      841 cgaggacggc ggcgccgtga ccgtgaccca ggacacctcc ctggaggacg gcaccctgat
      901 ctacaaggtg aagctccgcg gcaccaactt ccctcctgac ggccccgtaa tgcagaagaa
      961 gacaatgggc tgggaagcgt ccaccgagcg gttgtacccc gaggacggcg tgctgaaggg
     1021 cgacattaag atggccctgc gcctgaagga cggcggacgc tacctggcgg acttcaagac
     1081 cacctacaag gccaagaagc ccgtgcagat gcccggcgcc tacaacgtgg accgcaagtt
     1141 ggacatcacc tcccacaacg aggactacac cgtggtggaa cagtacgaac gctccgaggg
     1201 ccgccactcc accggcggca tggacgagct gtacaagatg gccccaaaga agaagcggaa
     1261 ggtcggtatc cacggagtcc cagcagccgg aagcggagcg acgaatttta gtctactgaa
     1321 acaagcggga gacgtggagg aaaaccctgg acctggagtg caggtggaaa ccatctcccc
     1381 aggagacggg cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat
     1441 gcttgaagat ggaaagaaag ttgattcctc ccgggacaga aacaagccct ttaagtttat
     1501 gctaggcaag caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg
     1561 tcagagagcc aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg
     1621 catcatccca ccacatgcca ctctcgtctt cgatgtggag cttctaaaac tggaatctgg
     1681 cggtggatcc ggagtcgacg gatttggtga tgtcggtgct cttgagagtt tgaggggaaa
     1741 tgcagatttg gcttacatcc tgagcatgga gccctgtggc cactgcctca ttatcaacaa
     1801 tgtgaacttc tgccgtgagt ccgggctccg cacccgcact ggctccaaca tcgactgtga
     1861 gaagttgcgg cgtcgcttct cctcgctgca tttcatggtg gaggtgaagg gcgacctgac
     1921 tgccaagaaa atggtgctgg ctttgctgga gctggcgcgg caggaccacg gtgctctgga
     1981 ctgctgcgtg gtggtcattc tctctcacgg ctgtcaggcc agccacctgc agttcccagg
     2041 ggctgtctac ggcacagatg gatgccctgt gtcggtcgag aagattgtga acatcttcaa
     2101 tgggaccagc tgccccagcc tgggagggaa gcccaagctc tttttcatcc aggcctgtgg
     2161 tggggagcag aaagaccatg ggtttgaggt ggcctccact tcccctgaag acgagtcccc
     2221 tggcagtaac cccgagccag atgccacccc gttccaggaa ggtttgagga ccttcgacca
     2281 gctggacgcc atatctagtt tgcccacacc cagtgacatc tttgtgtcct actctacttt
     2341 cccaggtttt gtttcctgga gggaccccaa gagtggctcc tggtacgttg agaccctgga
     2401 cgacatcttt gagcagtggg ctcactctga agacctgcag tccctcctgc ttagggtcgc
     2461 taatgctgtt tcggtgaaag ggatttataa acagatgcct ggttgcttta atttcctccg
     2521 gaaaaaactt ttctttaaaa catcagtcga ctatccgtac gacgtaccag actacgcact
     2581 cgactaacat atgcatggcc ggccgaattc ctgtgccttc tagttgccag ccatctgttg
     2641 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2701 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2761 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2821 cggtgggctc tatgataact tcgtatagca tacattatac gaagttatta attcgcggcc
     2881 gcaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc
     2941 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
     3001 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
     3061 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt
     3121 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg
     3181 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg
     3241 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag
     3301 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc
     3361 ctcttcctca tctccgggcc tttcgacctg cagcctgttg acaattaatc atcggcatag
     3421 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gcctttgtct
     3481 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     3541 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     3601 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     3661 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     3721 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     3781 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     3841 ggttatgtgt gggagggcta actagaacta tagtgagtcg tattacgtag atccagacat
     3901 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     3961 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     4021 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     4081 tttttaatta agcataactt cgtatagcat acattatacg aagttattga acattccctt
     4141 gatggtctca agctatgatc agaagacttt aattatatat tttcatccta taagcttaaa
     4201 taggaaagtt tcttcaacag gattacagtg tagctaccta catgctgaaa aatatagcct
     4261 ttaaatcatt tttatattat aactctgtat aatagagata agtccatttt ttaaaaatgt
     4321 tttccccaaa ccataaaacc ctatacaagt tgttctagta acaatacatg agaaagatgt
     4381 ctatgtagct gaaaataaaa tgacgtcaca agacaattgg tgtgtcattg actcttctat
     4441 tttgattttc ttttctgtgt aattcagtgg tttaatttga cattaaggga tacaagcctg
     4501 aattctagat tataattatt taatgaaata gagttcacat tctgaattga agaaaatact
     4561 tatagctttt gaaaagggac cttcaaggga atgtttagtt tcaat
//
