LOCUS Exported 8955 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Lentiviral expression of CDKN2A H123Q. ACCESSION . VERSION . KEYWORDS pHAGE-CDKN2A-H123Q SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8955) AUTHORS Ng PK, Li J, Jeong KJ, Shao S, Chen H, Tsang YH, Sengupta S, Wang Z, Bhavana VH, Tran R, Soewito S, Minussi DC, Moreno D, Kong K, Dogruluk T, Lu H, Gao J, Tokheim C, Zhou DC, Johnson AM, Zeng J, Ip CKM, Ju Z, Wester M, Yu S, Li Y, Vellano CP, Schultz N, Karchin R, Ding L, Lu Y, Cheung LWT, Chen K, Shaw KR, Meric-Bernstam F, Scott KL, Yi S, Sahni N, Liang H, Mills GB TITLE Systematic Functional Annotation of Somatic Mutations in Cancer. JOURNAL Cancer Cell. 2018 Mar 12;33(3):450-462.e10. doi: 10.1016/j.ccell.2018.01.021. PUBMED 29533785 REFERENCE 2 (bases 1 to 8955) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8955 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 129..246 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 294..1472 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 525..1463 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1420..1440 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 1492..1516 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(1986..2010) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter 2018..2036 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2061..2080) /label=T7 /note="T7 promoter, forward primer" promoter complement(2062..2080) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind complement(2086..2106) /gene="mutant version of attB" /label=attB4 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" CDS 2160..2201 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" misc_feature 2222..2789 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" misc_feature 2239..2791 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2383..2400) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2610..2629 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 2803..3402 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(2803..2822) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3299..3319 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 3416..4004 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3469..3489) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3887..3898) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 4079..4312 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(4431..4449) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4517..4621 /gene="bla" /label=AmpR promoter CDS 4622..5482 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(4840..4859) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5653..6241 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6142..6161 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6395..6412 /label=L4440 /note="L4440 vector, forward primer" primer_bind 6504..6523 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" protein_bind 6652..6673 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6688..6718 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6726..6742 /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 6731..6753 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6750..6766 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6750..6766 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6816..6835 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" LTR 7027..7660 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 7707..7832 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8329..8562 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8747..8791 /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" ORIGIN 1 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag 61 atccattcga ttagtgaacg gatctcgacg gtatcgccga attcacaaat ggcagtattc 121 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 181 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 241 aattttcggg tttattacag ggacagcaga gatccagttt ggactagtcg tgaggctccg 301 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg 361 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg 421 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg 481 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg 541 gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca 601 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt 661 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc 721 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata 781 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata 841 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg 901 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc 961 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg 1021 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt 1081 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc 1141 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag 1201 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct 1261 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc 1321 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct 1381 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc 1441 aaagtttttt tcttccattt caggtgtcgt gaagcggccc tgcagatatc aacaagtttg 1501 tacaaaaaag ttggcatgga gccggcggcg gggagcagca tggagccttc ggctgactgg 1561 ctggccacgg ccgcggcccg gggtcgggta gaggaggtgc gggcgctgct ggaggcgggg 1621 gcgctgccca acgcaccgaa tagttacggt cggaggccga tccaggtcat gatgatgggc 1681 agcgcccgag tggcggagct gctgctgctc cacggcgcgg agcccaactg cgccgacccc 1741 gccactctca cccgacccgt gcacgacgct gcccgggagg gcttcctgga cacgctggtg 1801 gtgctgcacc gggccggggc gcggctggac gtgcgcgatg cctggggccg tctgcccgtg 1861 gacctggctg aggagctggg ccaacgcgat gtcgcacggt acctgcgcgc ggctgcgggg 1921 ggcaccagag gcagtaacca tgcccgcata gatgccgcgg aaggtccctc agatatcccc 1981 gattgaccca actttcttgt acaaagtggt gatatccaat taaccctcac taaaggagta 2041 agtgttagat agtattgaat ccctatagtg agtcgtatta ccacccaact tttctataca 2101 aagtggttga tatccagcac agtggcggcc gctcgagtct agagggcccg cggttcgaag 2161 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gcgtaccggt tagtaatgag 2221 atccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt 2281 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac 2341 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc 2401 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa 2461 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg 2521 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg 2581 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc 2641 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat 2701 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg 2761 tggttttcct ttgaaaaaca cgatgataat atggccacac atatgaccga gtacaagccc 2821 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg 2881 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc 2941 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc 3001 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg 3061 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag 3121 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc 3181 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc 3241 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc 3301 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa 3361 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct aaatcgatag atcctaatca 3421 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 3481 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 3541 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 3601 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 3661 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 3721 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 3781 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg 3841 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 3901 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 3961 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctgaga tcctttaaga 4021 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg 4081 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc 4141 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 4201 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 4261 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt 4321 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag 4381 aggcccgggt taattaagga aagggctaga tcattcttga agacgaaagg gcctcgtgat 4441 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 4501 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 4561 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 4621 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 4681 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 4741 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 4801 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 4861 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 4921 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 4981 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 5041 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 5101 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 5161 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 5221 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 5281 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 5341 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 5401 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 5461 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 5521 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 5581 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 5641 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 5701 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 5761 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 5821 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 5881 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 5941 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 6001 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 6061 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 6121 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 6181 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 6241 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 6301 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 6361 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 6421 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagcaa 6481 gctcatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 6541 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccc 6601 gtggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 6661 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 6721 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgaattt 6781 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 6841 atcttatcat gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc 6901 caccccatac cctattacca ctgccaatta cctgtggttt catttactct aaacctgtga 6961 ttcctctgaa ttattttcat tttaaagaaa ttgtatttgt taaatatgta ctacaaactt 7021 agtagttgga agggctaatt cactcccaaa gaagacaaga tatccttgat ctgtggatct 7081 accacacaca aggctacttc cctgattagc agaactacac accagggcca ggggtcagat 7141 atccactgac ctttggatgg tgctacaagc tagtaccagt tgagccagat aaggtagaag 7201 aggccaataa aggagagaac accagcttgt tacaccctgt gagcctgcat gggatggatg 7261 acccggagag agaagtgtta gagtggaggt ttgacagccg cctagcattt catcacgtgg 7321 cccgagagct gcatccggag tacttcaaga actgctgata tcgagcttgc tacaagggac 7381 tttccgctgg ggactttcca gggaggcgtg gcctgggcgg gactggggag tggcgagccc 7441 tcagatcctg catataagca gctgcttttt gcctgtactg ggtctctctg gttagaccag 7501 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 7561 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 7621 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact 7681 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc 7741 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag 7801 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc 7861 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag 7921 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag 7981 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac 8041 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa 8101 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg 8161 cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg 8221 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac 8281 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt 8341 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt 8401 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat 8461 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag 8521 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc 8581 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct 8641 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac 8701 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga 8761 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct 8821 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt 8881 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac 8941 ccacctccca acccc //