LOCUS       sequence-328838-        5138 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express Venus fused to the N-terminus of the Bcl-2 family protein, 
            tBid with mutation to 2 positions in the BH3 domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5138)
  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 5138)
  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"; 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-tBid-2E-pEGFP-C1
FEATURES             Location/Qualifiers
     source          1..5138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        97..400
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        401..604
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     555..575
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      643..652
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(649..651,652..654,655..1365)
                     /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
                     ERVTAAGITLGMDELYK"
     primer_bind     complement(694..715)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(955..974)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    1786..1838
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1962..2083
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1999..2018)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2053..2072
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2090..2545)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2227..2248)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2439..2458
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2572..2676
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2678..3035
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2678..2869
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2701..2721)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2886..3021
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3070..3864
                     /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             3070..3864
                     /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(3124..3143)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3734..3753
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4052..4071)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4096..4143
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4472..5060
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4961..4980
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac
       61 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      121 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      181 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      241 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      301 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      361 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      421 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      481 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      541 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      601 agctggttta gtgaaccgtc agatccgcta gcgctaccgg tcgccaccat ggtgagcaag
      661 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      721 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
      781 ctgaagctga tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
      841 ctgggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
      901 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
      961 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1021 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1081 aactacaaca gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1141 aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag
     1201 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     1261 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggaacgc
     1321 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggagg ctccggcagc
     1381 caggccagcc gctccttcaa ccaaggaaga atagagccag attctgaaag tcaggaagaa
     1441 atcatccaca acgaagccag acatgaggcc caaataggcg atgagatgga ccacaacatc
     1501 cagcccacac tggtgagaca gctagccgca cagttcatga atggcagcct gtcggaggaa
     1561 gacaaaagga actgcctggc caaagccctt gatgaggtga agacagcctt ccccagagac
     1621 atggagaacg acaaggccat gctgataatg accatgctgt tggccaaaaa agtggccagt
     1681 cacgcaccat ctttgctccg tgatgtcttc cacacgactg tcaactttat taaccagaac
     1741 ctattctcct atgtgaggaa cttggttaga aacgagatgg actaactcga gctcaagctt
     1801 cgaattctgc agtcgacggt accgcgggcc cgggatccac cggatctaga taactgatca
     1861 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     1921 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     1981 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2041 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc
     2101 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     2161 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     2221 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     2281 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     2341 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     2401 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     2461 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     2521 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     2581 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2641 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     2701 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2761 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     2821 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     2881 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     2941 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     3001 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     3061 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     3121 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     3181 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     3241 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     3301 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     3361 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     3421 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     3481 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     3541 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     3601 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     3661 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     3721 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     3781 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     3841 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     3901 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     3961 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4021 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     4081 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     4141 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     4201 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     4261 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     4321 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4381 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4441 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4501 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4561 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4621 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtatcaccg cctacatacc
     4681 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4741 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4801 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4861 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4921 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4981 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5041 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5101 gctggccttt tgctcacatg ttctttcctg cgttatcc
//
