LOCUS       sequence-216870-        8533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of p16INK4A IRES H2B-mOrange2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8533)
  AUTHORS   Zeng H, Jorapur A, Shain AH, Lang UE, Torres R, Zhang Y, McNeal AS, 
            Botton T, Lin J, Donne M, Bastian IN, Yu R, North JP, Pincus L, 
            Ruben BS, Joseph NM, Yeh I, Bastian BC, Judson RL
  TITLE     Bi-allelic Loss of CDKN2A Initiates Melanoma Invasion via BRN2 
            Activation.
  JOURNAL   Cancer Cell. 2018 Jul 9;34(1):56-68.e9. doi: 
            10.1016/j.ccell.2018.05.014.
  PUBMED    29990501
REFERENCE   2  (bases 1 to 8533)
  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"; doi: 
            "10.1016/j.ccell.2018.05"; journalName: "Cancer Cell"; date: 
            "2018-07-9- 9"; volume: "34"; issue: "1"; pages: "56-68.e9"
COMMENT     Sequence Label: pHIV-INK4A-mOrange2
FEATURES             Location/Qualifiers
     source          1..8533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2624..3802
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2855..3793
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3750..3770
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    4310..4883
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4333..4885
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4477..4494)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4704..4723
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4889..5266
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      5279..5288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5285..5995
                     /codon_start=1
                     /product="photostable monomeric orange derivative of DsRed 
                     fluorescent protein (Shaner et al., 2008)"
                     /label=mOrange2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTAKLKVTKGGPLPFAWDILSPHFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNY
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GKIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6013..6029)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6014..6030)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6539..6719
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6781..7369)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6861..6880)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7540..8331,8332..8400))
                     /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     8163..8182
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8401..8505)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgctctagcg tgaggctccg gtgcccgtca
     2641 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
     2701 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
     2761 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
     2821 tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg
     2881 gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca
     2941 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg
     3001 cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg
     3061 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc
     3121 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa
     3181 tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc
     3241 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat
     3301 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg
     3361 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag
     3421 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga
     3481 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc
     3541 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg
     3601 gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga
     3661 gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc
     3721 cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt
     3781 tcttccattt caggtgtcgt gagcggccgc tgaactgaat tcatcgacgt taactattct
     3841 agagtaccca tggagccttc ggctgactgg ctggccacgg ccgcggcccg gggtcgggta
     3901 gaggaggtgc gggcgctgct ggaggcgggg gcgctgccca acgcaccgaa tagttacggt
     3961 cggaggccga tccaggtcat gatgatgggc agcgcccgag tggcggagct gctgctgctc
     4021 cacggcgcgg agcccaactg cgccgacccc gccactctca cccgacccgt gcacgacgct
     4081 gcccgggagg gcttcctgga cacgctggtg gtgctgcacc gggccggggc gcggctggac
     4141 gtgcgcgatg cctggggccg tctgcccgtg gacctggctg aggagctggg ccatcgcgat
     4201 gtcgcacggt acctgcgcgc ggctgcgggg ggcaccagag gcagtaacca tgcccgcata
     4261 gatgccgcgg aaggtccctc agacatcccc gattgagggc taggatccgc ccctctccct
     4321 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     4381 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     4441 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     4501 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     4561 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     4621 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     4681 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     4741 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtac acatgcttta
     4801 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     4861 tcctttgaaa aacacgatga taatatggat gccagagcca gcgaagtctg ctcccgcccc
     4921 gaaaaagggc tccaagaagg cggtgactaa ggcgcagaag aaaggcggca agaagcgcaa
     4981 gcgcagccgc aaggagagct attccatcta tgtgtacaag gttctgaagc aggtccaccc
     5041 tgacaccggc atttcgtcca aggccatggg catcatgaat tcgtttgtga acgacatttt
     5101 cgagcgcatc gcaggtgagg cttcccgcct ggcgcattac aacaagcgct cgaccatcac
     5161 ctccagggag atccagacgg ccgtgcgcct gctgctgcct ggggagttgg ccaagcacgc
     5221 cgtgtccgag ggtactaagg ccatcaccaa gtacaccagc gctaaggatc caccggtcgc
     5281 caccatggtg agcaagggcg aggagaataa catggccatc atcaaggagt tcatgcgctt
     5341 caaggtgcgc atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     5401 gggccgcccc tacgagggct ttcagaccgc taagctgaag gtgaccaagg gtggccccct
     5461 gcccttcgcc tgggacatcc tgtcccctca tttcacctac ggctccaagg cctacgtgaa
     5521 gcaccccgcc gacatccccg actacttcaa gctgtccttc cccgagggct tcaagtggga
     5581 gcgcgtgatg aactacgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     5641 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     5701 cgtgatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     5761 cggtgccctg aagggcaaga tcaagatgag gctgaagctg aaggacggcg gccactacac
     5821 ctccgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacat
     5881 cgtcgacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     5941 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagcggc
     6001 cgcgactcta gacgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac
     6061 aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga
     6121 ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc
     6181 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     6241 acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg
     6301 gcagaactac acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa
     6361 gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt
     6421 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag
     6481 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg
     6541 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     6601 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     6661 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     6721 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6781 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6841 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6901 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6961 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     7021 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7081 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7141 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7201 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     7261 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7321 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7381 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7441 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7501 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7561 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7621 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7681 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7741 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7801 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7861 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7921 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7981 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     8041 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     8101 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     8161 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8221 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     8281 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8341 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8401 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8461 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8521 agtgccacct gac
//
