LOCUS       sequence-216850-        7726 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Recombination template for replacing CDKN2A Exon2 with 
            Ef1alpha-H2B-mOrange2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7726)
  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 7726)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-H2B-mOrange2
FEATURES             Location/Qualifiers
     source          1..7726
                     /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"
     CDS             4372..4749
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      4762..4771
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4768..5478
                     /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"
     polyA_signal    5600..5721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5637..5656)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5691..5710
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
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 gaaggacagt 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 ctctctgact 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 gctggtttag tgaaccgtca gatccgctag cgctaccgga
     4321 ctcagatctc gagctcaagc ttcgaattct gcagtcgacg gtaccgccac catgccagag
     4381 ccagcgaagt ctgctcccgc cccgaaaaag ggctccaaga aggcggtgac taaggcgcag
     4441 aagaaaggcg gcaagaagcg caagcgcagc cgcaaggaga gctattccat ctatgtgtac
     4501 aaggttctga agcaggtcca ccctgacacc ggcatttcgt ccaaggccat gggcatcatg
     4561 aattcgtttg tgaacgacat tttcgagcgc atcgcaggtg aggcttcccg cctggcgcat
     4621 tacaacaagc gctcgaccat cacctccagg gagatccaga cggccgtgcg cctgctgctg
     4681 cctggggagt tggccaagca cgccgtgtcc gagggtacta aggccatcac caagtacacc
     4741 agcgctaagg atccaccggt cgccaccatg gtgagcaagg gcgaggagaa taacatggcc
     4801 atcatcaagg agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag
     4861 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gctttcagac cgctaagctg
     4921 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcatttcacc
     4981 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt caagctgtcc
     5041 ttccccgagg gcttcaagtg ggagcgcgtg atgaactacg aggacggcgg cgtggtgacc
     5101 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     5161 accaacttcc cctccgacgg ccccgtgatg cagaagaaga ccatgggctg ggaggcctcc
     5221 tccgagcgga tgtaccccga ggacggtgcc ctgaagggca agatcaagat gaggctgaag
     5281 ctgaaggacg gcggccacta cacctccgag gtcaagacca cctacaaggc caagaagccc
     5341 gtgcagctgc ccggcgccta catcgtcgac atcaagttgg acatcacctc ccacaacgag
     5401 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     5461 gacgagctgt acaagtaagc ggccgcgact ctagatcata atcagccata ccacatttgt
     5521 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     5581 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     5641 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5701 caaactcatc aatgtatctt agatcctcag gtgaggactg atgatctgag aatttgtacc
     5761 ctgagagctt ccaaagctca gagcattcat tttccagcac agaaagttca gcccgggaga
     5821 ccagtctccg gtcttgcctc agctcacgcg ccaatcggtg ggacggcctg agtctcccta
     5881 tcgccctgcc ccgccagggc ggcaaatggg aaataatccc gaaatggact tgcgcacgtg
     5941 aaagcccatt ttgtacatta tacttcccaa agcataccac cacccaaaca cctaccctct
     6001 gctagttcaa ggcctagact gcggagcaat gaagactcaa gaggctagag gtctagtgcc
     6061 ccctcttcct ccaaactagg gccagttgca tccacttacc aggtctgttt cctcatttgc
     6121 ataccaagct ggctggacca acctcaggat ttccaaaccc aattgtgcgt ggcatcatct
     6181 ggagatctct cgatctcggc tcttctgcac aactcaacta atctgaccct cctcagctaa
     6241 tctgaccctc cgctttatgc ggtagagttt tccagagctg ccccaggggg ttctggggac
     6301 atcaggacca agacttcgct gaccctggca gtctgtgcac cggagttggc tcctttccct
     6361 cttaaacttg tgcaagagat cgctgagcga tgaaggtaga attatggtcc tccttgccct
     6421 tgcctttcct ttttgtgatc tcaaagcatc ctccctccgc ccccattcca tggccccagt
     6481 tccctactcc cacagctgtc tgctgaaact gccaacatta ctcaattgtt tctgggggga
     6541 ggaacatttt tttttgaaac aaaatagata tatgaaacag tacacgggaa ttaacacgaa
     6601 tatttaaggt aaaacatgac cttgaagatt atgaaatcca tcttattttg gcccagaacg
     6661 ggggcattgg gctccttggg ccatagggga gctggggagg acagggtgaa gagttagctc
     6721 taagccctct gcttggagat gctgtaaata cagaacgcaa aatcaccttc gaagttaaag
     6781 acgcgaagtt cttctttact cggcccctcc tcccctcccc cccgccaatt ccctccagtt
     6841 acagctagca tccaggtccc gggaggtgaa gaaggagact tcggctccag ttacagctag
     6901 catccgggtc ccgatttaga aggagctgcc aattacagcg cggttccagg gctgagcaaa
     6961 aagcctgagg agccaagtgg gagagggagt aaaactactg aattgggcca caagcaaatg
     7021 aataaactga acgactctta accaaaccta atatatttaa tccaaacaca caagtctttc
     7081 atttcttccc tcctcccttc cttctcttac tccccaacac cccctcttca agcacaatta
     7141 attatatggt tagattctac tgcgtgatca gccctgttct aggtggtggg cacgccaagg
     7201 tgaatgagac caaacaagag tcttgccctc atggggttta catttggaga cagagtcgat
     7261 ctgttgccca acctggagtg cagtggcgcg atcacagctc actgcagcct caaactccct
     7321 ggctcaaggg gttctcccac ctgagcctcc cgactagctg ggaccacagg tgcacgccac
     7381 gacgcctggg tttgtttgtt tgtttaatag agacgaaggt ctcaccatgt tatctgggct
     7441 caagcgatca tcccccctcc tcctcctaaa gtactgggat tacagtccca agctatcttg
     7501 cccgacctgg gaaacagacg ttaaggaaga taacaatcta ttttcagaga gcgagtttat
     7561 aaaaccaatg caatgggtaa atatgaagtg tgaataggag gagaagctaa agagtggtcg
     7621 gagaatctaa tgcaagctac gggagaaaga aactcaagtg caaatgctgc ctcaggaata
     7681 aacgtaaaaa gagactttca agtgcaaatg ctccctcagg aataaa
//
