LOCUS       sequence-329014-        5492 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of mCerulean3 linked to Venus by 39 amino acid 
            linker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5492)
  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 5492)
  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: mCerulean3-39aa-Venus-pEGFP-C3
FEATURES             Location/Qualifiers
     source          1..5492
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        106..409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        410..613
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     564..584
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      652..661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(658..660,661..663,664..1374)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      1486..1495
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1492..1494,1495..1497,1498..2208)
                     /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"
     polyA_signal    2325..2446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2362..2381)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2416..2435
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2453..2908)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2590..2611)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2802..2821
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2935..3039
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3041..3398
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3041..3232
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3064..3084)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3249..3384
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3433..4227
                     /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             3433..4227
                     /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(3487..3506)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4097..4116
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4415..4434)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4459..4506
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4835..5423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5324..5343
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta
       61 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      121 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      181 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      241 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      301 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      361 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      421 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      481 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      541 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      601 tataagcaga gctggtttag tgaaccgtca gatccgctag cgctaccggt cgccaccatg
      661 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      721 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      781 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      841 gtgaccaccc tgagctgggg cgtgcagtgc ttcgcccgct accccgacca catgaagcag
      901 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      961 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1021 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1081 ctggagtaca acgccatcca cggcaacgtc tatatcaccg ccgacaagca gaagaacggc
     1141 atcaaggcca acttcggcct caactgcaac atcgaggacg gcagcgtgca gctcgccgac
     1201 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1261 ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1321 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga
     1381 ctcagatctc gaggttcggg cctgcgctcg ccacccgtag caactgctcg aggttcgggc
     1441 ctgcgctcgc cacccgtagc aactgctcga ggaggaggac cggtcgccac catggtgagc
     1501 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1561 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1621 accctgaagc tgatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1681 accctgggct acggcctgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
     1741 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1801 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1861 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1921 tacaactaca acagccacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag
     1981 gccaacttca agatccgcca caacatcgag gacggcggcg tgcagctcgc cgaccactac
     2041 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2101 taccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggaa
     2161 cgcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtc cggactcaga
     2221 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     2281 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     2341 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2401 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta
     2461 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     2521 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     2581 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     2641 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     2701 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     2761 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     2821 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     2881 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2941 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3001 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     3061 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3121 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3181 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3241 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3301 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     3361 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     3421 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3481 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3541 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3601 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3661 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3721 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3781 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3841 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3901 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3961 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4021 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4081 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4141 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4201 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4261 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4321 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4381 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     4441 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     4501 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     4561 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     4621 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     4681 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4741 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4801 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4861 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4921 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4981 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5041 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5101 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5161 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5221 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5281 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5341 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5401 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5461 tttgctggcc ttttgctcac atgttctttc ct
//
