LOCUS       sequence-214270-        7370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombination template for replacing CDKN2A Exon2 with CMV-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7370)
  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 7370)
  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: pHDR-CDKN2A-Ex2-CMV-EGFP
FEATURES             Location/Qualifiers
     source          1..7370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(24..612)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(104..123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(783..1574,1575..1643))
                     /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     1406..1425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1644..1748)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        3776..4079
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4080..4283
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4234..4254
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4280..4304
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(4403..4405,4406..4408,4409..5122)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4403..4405,4406..4408,4409..5119)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4448..4469)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4709..4728)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5056..5077
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5130..5354
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gtcgactcga gaattccggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
       61 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      121 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
      181 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
      241 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
      301 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
      361 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
      421 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
      481 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
      541 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
      601 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
      661 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
      721 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
      781 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
      841 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
      901 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
      961 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     1021 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     1081 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     1141 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     1201 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     1261 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     1321 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     1381 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     1441 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     1501 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     1561 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     1621 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     1681 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     1741 gttccgcgca catttccatc gatacgcgtg cttttaaata tttacatatg ggttactttt
     1801 tagatttttc atactatgaa atataagaaa aacctttaag gctagtttta tgaccaagac
     1861 gaaggacttc attgaataca caaaacaata aatatactgc aacattttgt ctttcttttt
     1921 gtagctgcaa tttggtttgc ttatactttc tctttgtctc tttgaaaact gagtcagttt
     1981 cactttctca ggacaggatt taataaccat aatataattt agtataattc cttgatttag
     2041 gcaaattatg caatttgtgt ttagtatgaa atgtacctaa aaataagtaa ctcctcttta
     2101 acaccaccat cctcaaacta atataacaaa taacagttat cctaaaataa attgtctact
     2161 tccaccatgc agcactcaaa ttttaaggtt gctatgactg cagacagtat tttaaaattc
     2221 ctctctggaa atggctttgt ttccaagatg atttaggaac caaagaggtg accatctctt
     2281 gtttaatgaa ctctcaaatc ataaacctgg gaagtgtttt agtttcctac tgctgctgtt
     2341 acaaattatc acaaatgtgt tagctaaaac aaacacaaaa ttattatttt acagttctag
     2401 agatcagaag tcaaaaatgg gtccacaagg tttcattcct tttggaaact ctaaggggca
     2461 atctgtttcc ttgtcttttc cagcttctag tgaccatcaa attccttggc tcatggtctc
     2521 tgtattttct ctgtggcctg tgcttccatt cttgtatctt ctctctggct gtgaccctct
     2581 aataaaaaca cttggggtta tgttgggccc accctgaaaa ttctggataa tctccctcaa
     2641 gaccattaat taaatcacat ctgcaaagcc tcttttgcca cataagttaa tgtattaaaa
     2701 gtttttgagg attaggacat agacattggg ggtggggggg cattattcag cctaccacag
     2761 gaaggaattt tagggttaat taaactagcc ttcttatttt atacttgaag aaattgaagt
     2821 tttggaattg gagagcatta tgctaaatga aataagccaa acacagaaag acaaatatca
     2881 catgttctca cttatctgtg aaatataaaa caattacatt cttagcagta aagagtagaa
     2941 tggtggttac tagagctggg gggtgggagg aatggggaga tggtaatcaa gatataaagc
     3001 ctcagttaag atgggaggaa taagtttgat tgtttttttt gagatgtgtt tcatagcatg
     3061 atgaatatag ctaaatagta aatcccaaat gctctcattt gacaaaaatg tcaaatattt
     3121 gagatgatgg ataggttact tagcttgact taataattcc ccattgtgtt caaagatcat
     3181 aacttcatat tgtaccacat aaatatatac aactgtacta tcccaatata taattttaaa
     3241 actaatataa tgaaaaagaa attgaagttc aacattccca gaagctaagt gtaacttaaa
     3301 agttttgtga gaatttgttt taacaaacaa acaagttttc tctttttaac aattaccaca
     3361 ttctgcgctt ggatatacag cagtgaacaa aaaaaaaaaa aaaaatctcc aggcctaaca
     3421 taatttcagg aagaaatttc agtagttgta tctcagggga aatacaggaa gttagcctgg
     3481 agtaaaagtc agtctgtccc tgcccctttg ctattttgcc cgtgcctcac agtgctctct
     3541 gcctgtgacg acagctccgc agaagttcgg aggatataat ggaattcatt gtgtactgaa
     3601 gaatggatag agaactcaag aaggaaattg gaaactggaa gcaaatgtag gggtaattag
     3661 acacctgggg cttgtgtggg ggtctgcttg gcggtgaggg ggctctacac aagcttcctt
     3721 tccgtcatgc cggcccccac cctggctctg accattctgt tctctctggc agccgcgtta
     3781 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     3841 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     3901 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     3961 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     4021 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     4081 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     4141 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     4201 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     4261 gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
     4321 cacgctgttt tgacctccat agaagacacc gactctagag gatccactag tccagtgtgg
     4381 tggaattcat ttaaatgcta gcatggtgag caagggcgag gagctgttca ccggggtggt
     4441 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     4501 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     4561 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     4621 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     4681 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     4741 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     4801 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     4861 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     4921 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     4981 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     5041 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     5101 catggacgag ctgtacaagt gagcggccgc tgtgccttct agttgccagc catctgttgt
     5161 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     5221 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     5281 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     5341 ggtgggctct atggtgattg aatgagatcc tcaggtgagg actgatgatc tgagaatttg
     5401 taccctgaga gcttccaaag ctcagagcat tcattttcca gcacagaaag ttcagcccgg
     5461 gagaccagtc tccggtcttg cctcagctca cgcgccaatc ggtgggacgg cctgagtctc
     5521 cctatcgccc tgccccgcca gggcggcaaa tgggaaataa tcccgaaatg gacttgcgca
     5581 cgtgaaagcc cattttgtac attatacttc ccaaagcata ccaccaccca aacacctacc
     5641 ctctgctagt tcaaggccta gactgcggag caatgaagac tcaagaggct agaggtctag
     5701 tgccccctct tcctccaaac tagggccagt tgcatccact taccaggtct gtttcctcat
     5761 ttgcatacca agctggctgg accaacctca ggatttccaa acccaattgt gcgtggcatc
     5821 atctggagat ctctcgatct cggctcttct gcacaactca actaatctga ccctcctcag
     5881 ctaatctgac cctccgcttt atgcggtaga gttttccaga gctgccccag ggggttctgg
     5941 ggacatcagg accaagactt cgctgaccct ggcagtctgt gcaccggagt tggctccttt
     6001 ccctcttaaa cttgtgcaag agatcgctga gcgatgaagg tagaattatg gtcctccttg
     6061 cccttgcctt tcctttttgt gatctcaaag catcctccct ccgcccccat tccatggccc
     6121 cagttcccta ctcccacagc tgtctgctga aactgccaac attactcaat tgtttctggg
     6181 gggaggaaca tttttttttg aaacaaaata gatatatgaa acagtacacg ggaattaaca
     6241 cgaatattta aggtaaaaca tgaccttgaa gattatgaaa tccatcttat tttggcccag
     6301 aacgggggca ttgggctcct tgggccatag gggagctggg gaggacaggg tgaagagtta
     6361 gctctaagcc ctctgcttgg agatgctgta aatacagaac gcaaaatcac cttcgaagtt
     6421 aaagacgcga agttcttctt tactcggccc ctcctcccct cccccccgcc aattccctcc
     6481 agttacagct agcatccagg tcccgggagg tgaagaagga gacttcggct ccagttacag
     6541 ctagcatccg ggtcccgatt tagaaggagc tgccaattac agcgcggttc cagggctgag
     6601 caaaaagcct gaggagccaa gtgggagagg gagtaaaact actgaattgg gccacaagca
     6661 aatgaataaa ctgaacgact cttaaccaaa cctaatatat ttaatccaaa cacacaagtc
     6721 tttcatttct tccctcctcc cttccttctc ttactcccca acaccccctc ttcaagcaca
     6781 attaattata tggttagatt ctactgcgtg atcagccctg ttctaggtgg tgggcacgcc
     6841 aaggtgaatg agaccaaaca agagtcttgc cctcatgggg tttacatttg gagacagagt
     6901 cgatctgttg cccaacctgg agtgcagtgg cgcgatcaca gctcactgca gcctcaaact
     6961 ccctggctca aggggttctc ccacctgagc ctcccgacta gctgggacca caggtgcacg
     7021 ccacgacgcc tgggtttgtt tgtttgttta atagagacga aggtctcacc atgttatctg
     7081 ggctcaagcg atcgtccccc ctcctcctcc taaagtactg ggattacagt cccaagctat
     7141 ctcgcccgac ctgggaaaca gacgttaagg aagataacaa tctattttca gagagcgagt
     7201 ttataaaacc aatgcaatgg gtaaatatga agtgtgaata ggaggagaag ctaaagagtg
     7261 gtcggagaat ctaatgcaag ctacgggaga aagaaactca agtgcaaatg ctgcctcagg
     7321 aataaacgta aaaagagact ttcaagtgca aatgctccct caggaataaa
//
