LOCUS       sequence-329007-        5300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Venus fused to the N-terminus of the Bcl-2 family protein, 
            Puma with mutation in BH3 domain to disrupt binding anti-apoptotic 
            proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5300)
  AUTHORS   Osterlund EJ, Hirmiz N, Pemberton JM, Nougarede A, Liu Q, Leber B, 
            Fang Q, Andrews DW
  TITLE     Efficacy and specificity of inhibitors of BCL-2 family protein 
            interactions assessed by affinity measurements in live cells.
  JOURNAL   Sci Adv. 2022 Apr 22;8(16):eabm7375. doi: 10.1126/sciadv.abm7375. 
            Epub 2022 Apr 20.
  PUBMED    35442739
REFERENCE   2  (bases 1 to 5300)
  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"; journalName: "Sci Adv."; 
            date: "2022-04-22"; volume: "8(16):eabm7375. doi"; pages: " 
            10.1126/sciadv.abm7375. Epub 2022 Apr 20"
COMMENT     Sequence Label: Venus-Puma-4E-pEGFP-C1
FEATURES             Location/Qualifiers
     source          1..5300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        52..355
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        356..559
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     510..530
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      598..607
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(604..606,607..609,610..1317)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     ERVTAAGITLGMDELY"
     primer_bind     complement(649..670)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(910..929)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    1903..1955
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2079..2200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2116..2135)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2170..2189
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2234..2662)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2344..2365)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2556..2575
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2689..2793
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2795..3152
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2795..2986
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2818..2838)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3003..3138
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3187..3981
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3187..3981
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3241..3260)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3851..3870
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4169..4188)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4213..4260
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4589..5177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5078..5097
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
       61 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      121 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      181 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      241 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      301 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      361 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      421 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      481 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      541 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc
      601 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
      661 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
      721 tacggcaagc tgaccctgaa gctgatctgc accaccggca agctgcccgt gccctggccc
      781 accctcgtga ccaccctggg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg
      841 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
      901 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
      961 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1021 cacaagctgg agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag
     1081 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
     1141 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1201 cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1261 gtcctgctgg aacgcgtgac cgccgccggg atcactctcg gcatggacga gctgtacagt
     1321 atggcacgcg cacgtcagga aggcagttca cctgaaccag tggaaggact ggcccgggat
     1381 gggccgcgtc catttccgct tgggcgcctg gttccgtcag ccgtctcttg cggactgtgc
     1441 gagccgggtc tggctgccgc accggctgca cccaccttac tgcccgccgc ttacctttgc
     1501 gcgcctacgg ctcccccagc agtaacggcc gccctgggag ggagccgttg gccgggcggc
     1561 cctcgtagcc gtccccgggg tccacgcccg gatggtccac agccaagtct gtcactggct
     1621 gaacaacatc ttgaatctcc cgtgccatca gcaccaggag ctctggctgg tggcccgaca
     1681 caagcagcac cgggggttcg gggcgaagaa gaacagtggg cccgggaaga aggtgcgcag
     1741 gaacgtcgcg aggccgatga tgagaacgcc cagtatgagc gtcgccgcca ggaggagcag
     1801 cagcgtcatc gcccctctcc ttggcgcgta ctttacaacc tgattatggg cttactgccc
     1861 ttaccgcgtg gccaccgggc tcccgagatg gaacccaatt aactcgagct caagcttcga
     1921 attctgcagt cgacggtacc gcgggcccgg gatccaccgg atctagataa ctgatcataa
     1981 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     2041 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     2101 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     2161 attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt
     2221 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     2281 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     2341 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     2401 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     2461 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     2521 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     2581 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     2641 aatgcgccgc tacagggcgt gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2701 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2761 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     2821 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2881 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2941 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3001 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3061 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3121 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     3181 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3241 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3301 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3361 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3421 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3481 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3541 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3601 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3661 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     3721 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     3781 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     3841 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     3901 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     3961 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4021 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4081 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4141 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     4201 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
     4261 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
     4321 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
     4381 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
     4441 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4501 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4561 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4621 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4681 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4741 tagtgtagcc gtagttaggc caccacttca agaactctgt atcaccgcct acatacctcg
     4801 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4861 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4921 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4981 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5041 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5101 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5161 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5221 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5281 ccgccatgca ttagttatta
//
