LOCUS       sequence-270914-        5257 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor vector to knock in P2A-mCherry to the C-terminus of endogenous
            human p16INK4A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5257)
  AUTHORS   Zhang Y, Hyle J, Wright S, Shao Y, Zhao X, Zhang H, Li C
  TITLE     A cis-element within the ARF locus mediates repression of p16 
            (INK4A) expression via long-range chromatin interactions.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Dec 9. pii: 1909720116. doi: 
            10.1073/pnas.1909720116.
  PUBMED    31818950
REFERENCE   2  (bases 1 to 5257)
  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: "Proc Natl 
            Acad Sci U S A. 2019 Dec 9. pii: 1909720116. doi: 
            10.1073/pnas.1909720116."
COMMENT     Sequence Label: pKnockinDonor-human-p16INK4A-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..5257
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             874..930
                     /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             931..1641
                     /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(1076..1094)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1344..1363
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2443..2462)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2444..2462)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2472..2489)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2472..2488)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2481..2503)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2630..3085
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2717..2736)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2927..2948
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3111..3215
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3216..3284,3285..4076)
                     /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(3434..3453)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4247..4835
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4736..4755
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4989..5006
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5123..5144
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5159..5164,5165..5182,5183..5189)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5197..5213
                     /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     5202..5224
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5221..5237
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5221..5237
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     join(5256..5257,1..19)
                     /label=T3
                     /note="T3 promoter, forward primer"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggacgattga aagaaccaga gaggcgagaa
       61 gctaaagagt ggtcggagaa tctaatgcaa gctacgggag aaagaaactc aagtgcaaat
      121 gctgcctcag gaataaacgt aaaaagagac tttcaagtgc aaatgctccc tcaggaataa
      181 aataatcttg agactctcaa gtgtaaatgc tgcctcggga gaaccgaacg gcgagctgga
      241 gcccatacgc aacgagatta gagaggaagg cagaagccag agcacatgaa taaatgagca
      301 tccattttgt ttcagaaatg atcggaaacc atttgtgggt ttgtagaagc aggcatgcgt
      361 agggaagcta cgggattccg ccgaggagcg ccagagcctg aggcgccctt tggttatcgc
      421 aagctggctg gctcactccg caccaggtgc aaaagatgcc tggggatgcg ggaagggaaa
      481 ggccacatct tcacgccttc gcgcctggca ttgtgagcaa ccactgagac tcattatata
      541 acactcgttt tcttcttgca accctgcggg ccgcgcggtc gcgctttctc tgccctccgc
      601 cgggtggacc tggagcgctt gagcggtcgg cgcgcctgga gcagccaggc gggcagtgga
      661 ctagctgctg gaccagggag gtgtgggaga gcggtggcgg cgggtacatg cacgtgaagc
      721 cattgcgaga actttatcca taagtatttc aatgccggta gggacggcaa gagaggaggg
      781 cgggatgtgc cacacatctt tgacctcagg tttctaacgc ctgttttctt tctgccctct
      841 gcagacatcc ccgatttaat taacggaagc ggagctacta acttctctct gttaaagcaa
      901 gcaggagacg tggaagaaaa ccccggtccc atggtgagca agggcgagga ggataacatg
      961 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     1021 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     1081 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     1141 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     1201 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     1261 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     1321 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     1381 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     1441 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     1501 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac
     1561 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     1621 atggacgagc tgtacaagtg aaagaaccag agaggctctg agaaacctcg ggaaacttag
     1681 atcatcagtc accgaaggtc ctacagggct acaactgccc ccgccacaac ccaccccgct
     1741 ttcgtagttt tcatttagaa aatagagctt ttaaaaatgt cctgcctttt aacgtagata
     1801 tatgccttcc cccactaccg taaatgtcca tttatatcat tttttatata ttcttataaa
     1861 aatgtaaaaa agaaaaacac cgcttctgcc ttttcactgt gttggagttt tctggagtga
     1921 gcactcacgc cctaagcgca cattcatgtg ggcatttctt gcgagcctcg cagcctccgg
     1981 aagctgtcga cttcatgaca agcattttgt gaactaggga agctcagggg ggttactggc
     2041 ttctcttgag tcacactgct agcaaatggc agaaccaaag ctcaaataaa aataaaataa
     2101 ttttcattca ttcactcatt tattgtcaac atttattgag cacctattac aacaatttca
     2161 tcgcatggaa gacagcatcg tttctgacac tgttgtttca tgtatctctt agaaaaacgc
     2221 tgctattaga catctaacac tatttatctt gaggtgataa aatatcaaaa gccgtgtctc
     2281 aagatcgatg aaatgcggtt aaaatgatga atagaaactc tagggggacc tcatatcgat
     2341 agactcgaga ctggcacatc tggagatccg tatttatccg gcttcccctt ccagatcacg
     2401 cgaggtttgg ggcctctctg gttctttcaa tcggccaatt cgccctatag tgagtcgtat
     2461 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     2521 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     2581 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     2641 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2701 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2761 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2821 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2881 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2941 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3001 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3061 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3121 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3181 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3241 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3301 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3361 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3421 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3481 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3541 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3601 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3661 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3721 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3781 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3841 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3901 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3961 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4021 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4081 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4141 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4201 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4261 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4321 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4381 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4441 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4501 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4561 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4621 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4681 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4741 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4801 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4861 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4921 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4981 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     5041 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     5101 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     5161 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     5221 caggaaacag ctatgaccat gattacgcca agcgcgc
//
