LOCUS       sequence-211416-        8540 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 8540)
  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 8540)
  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..8540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..69)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        241..620
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        622..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     775..795
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             838..1018
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1065..1190
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1689..1922
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2107..2151
                     /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    2449..2566
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2630..3808
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2861..3799
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3756..3776
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    4316..4889
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4339..4891
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4483..4500)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4710..4729
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4895..5272
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      5285..5294
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5291..6001
                     /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(6020..6036)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6021..6037)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6546..6726
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6788..7376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6868..6887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7547..8338,8339..8407))
                     /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     8170..8189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8408..8512)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
       61 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
      121 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
      181 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
      241 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      301 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      361 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      421 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      481 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      541 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      841 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      901 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      961 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     1021 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
     1081 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
     1141 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
     1201 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1261 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1321 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1381 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1441 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1501 acaaaagtaa gaccaccgca cagcaagcgg ccggccgcgc tgatcttcag acctggagga
     1561 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     1621 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     1681 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     1741 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     1801 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     1861 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     1921 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     1981 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     2041 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     2101 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     2161 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     2221 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     2281 tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga
     2341 atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg
     2401 gcactgcgtg cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa
     2461 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     2521 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     2581 ggacagcaga gatccagttt ggttagtacc gggcccgctc tagccgtgag gctccggtgc
     2641 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     2701 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     2761 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     2821 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
     2881 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg
     2941 gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag
     3001 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
     3061 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc
     3121 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct
     3181 tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga
     3241 cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc
     3301 gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc
     3361 gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
     3421 ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc
     3481 gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt
     3541 cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag
     3601 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca
     3661 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga
     3721 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag
     3781 tttttttctt ccatttcagg tgtcgtgagc ggccgctgaa ctgaattcat cgacgttaac
     3841 tattctagag tacccatgga gccttcggct gactggctgg ccacggccgc ggcccggggt
     3901 cgggtagagg aggtgcgggc gctgctggag gcgggggcgc tgcccaacgc accgaatagt
     3961 tacggtcgga ggccgatcca ggtcatgatg atgggcagcg cccgagtggc ggagctgctg
     4021 ctgctccacg gcgcggagcc caactgcgcc gaccccgcca ctctcacccg acccgtgcac
     4081 gacgctgccc gggagggctt cctggacacg ctggtggtgc tgcaccgggc cggggcgcgg
     4141 ctggacgtgc gcgatgcctg gggccgtctg cccgtggacc tggctgagga gctgggccat
     4201 cgcgatgtcg cacggtacct gcgcgcggct gcggggggca ccagaggcag taaccatgcc
     4261 cgcatagatg ccgcggaagg tccctcagac atccccgatt gagggctagg atccgcccct
     4321 ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
     4381 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
     4441 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
     4501 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
     4561 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
     4621 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
     4681 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
     4741 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat
     4801 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
     4861 tggttttcct ttgaaaaaca cgatgataat atggatgcca gagccagcga agtctgctcc
     4921 cgccccgaaa aagggctcca agaaggcggt gactaaggcg cagaagaaag gcggcaagaa
     4981 gcgcaagcgc agccgcaagg agagctattc catctatgtg tacaaggttc tgaagcaggt
     5041 ccaccctgac accggcattt cgtccaaggc catgggcatc atgaattcgt ttgtgaacga
     5101 cattttcgag cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca agcgctcgac
     5161 catcacctcc agggagatcc agacggccgt gcgcctgctg ctgcctgggg agttggccaa
     5221 gcacgccgtg tccgagggta ctaaggccat caccaagtac accagcgcta aggatccacc
     5281 ggtcgccacc atggtgagca agggcgagga gaataacatg gccatcatca aggagttcat
     5341 gcgcttcaag gtgcgcatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     5401 gggcgagggc cgcccctacg agggctttca gaccgctaag ctgaaggtga ccaagggtgg
     5461 ccccctgccc ttcgcctggg acatcctgtc ccctcatttc acctacggct ccaaggccta
     5521 cgtgaagcac cccgccgaca tccccgacta cttcaagctg tccttccccg agggcttcaa
     5581 gtgggagcgc gtgatgaact acgaggacgg cggcgtggtg accgtgaccc aggactcctc
     5641 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     5701 cggccccgtg atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     5761 cgaggacggt gccctgaagg gcaagatcaa gatgaggctg aagctgaagg acggcggcca
     5821 ctacacctcc gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     5881 ctacatcgtc gacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     5941 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta
     6001 agcggccgcg actctagatc gataccgtcg acctcgatcg agacctagaa aaacatggag
     6061 caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag
     6121 aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca
     6181 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     6241 actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc
     6301 ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt
     6361 gctacaagct agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca
     6421 cccgcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag
     6481 agtggaggtt tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggact
     6541 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     6601 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     6661 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     6721 ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6781 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6841 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6901 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6961 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     7021 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     7081 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     7141 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     7201 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     7261 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     7321 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     7381 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     7441 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     7501 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     7561 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     7621 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     7681 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7741 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7801 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7861 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7921 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7981 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     8041 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     8101 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     8161 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     8221 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     8281 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     8341 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     8401 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     8461 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     8521 cccgaaaagt gccacctgac
//
