LOCUS       sequence-96973-e        5950 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Co-expresses two cyan fluorescent protein variants, a short 
            fluorescence lifetime CFP variant in the nucleus and a CFP variant 
            with a long lifetime (mTurquoise2) in the cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5950)
  AUTHORS   Joosen L, Hink MA, Gadella TW Jr, Goedhart J
  TITLE     Effect of fixation procedures on the fluorescence lifetimes of 
            Aequorea victoria derived fluorescent proteins.
  JOURNAL   J Microsc. 2014 Sep 1. doi: 10.1111/jmi.12168.
  PUBMED    25179491
REFERENCE   2  (bases 1 to 5950)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Microsc. 
            2014 Sep 1. doi: 10.1111/jmi.12168."
COMMENT     Sequence Label: pH2B-SCFP1-2A-mTq2-NES
FEATURES             Location/Qualifiers
     source          1..5950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             657..1034
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1047..1056
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1053..1055,1056..1058,1059..1769)
                     /codon_start=1
                     /product="monomeric optimized variant of ECFP (Kremers et 
                     al., 2006)"
                     /label=SCFP3A
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1776..1829
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      1845..1854
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1851..1853,1854..1856,1857..2564)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     polyA_signal    2738..2859
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2775..2794)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2829..2848
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2866..3321)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3003..3024)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3215..3234
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3348..3452
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3454..3811
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3454..3645
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3477..3497)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3662..3797
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3846..4640
                     /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             3846..4640
                     /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(3900..3919)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4510..4529
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4828..4847)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4872..4919
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5248..5836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5737..5756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gccaccatgc
      661 cagagccagc gaagtctgct cccgccccga aaaagggctc caagaaggcg gtgactaagg
      721 cgcagaagaa aggcggcaag aagcgcaagc gcagccgcaa ggagagctat tccatctatg
      781 tgtacaaggt tctgaagcag gtccaccctg acaccggcat ttcgtccaag gccatgggca
      841 tcatgaattc gtttgtgaac gacattttcg agcgcatcgc aggtgaggct tcccgcctgg
      901 cgcattacaa caagcgctcg accatcacct ccagggagat ccagacggcc gtgcgcctgc
      961 tgctgcctgg ggagttggcc aagcacgccg tgtccgaggg tactaaggcc atcaccaagt
     1021 acaccagcgc taaggatcca ccggtcgcca ccatggtgag caagggcgag gagctgttca
     1081 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1141 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1201 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tggggcctgc
     1261 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1321 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1381 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1441 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac atcagccaca
     1501 acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc
     1561 acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1621 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     1681 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1741 tcactctcgg catggacgag ctgtacaagt ccggagaggg cagaggaagt cttctaacat
     1801 gcggtgacgt ggaggagaat cccggccctg gggatccacc ggtcgccacc atggtgagca
     1861 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1921 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1981 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2041 ccctgtcctg gggcgtgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact
     2101 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2161 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2221 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2281 acaactactt tagcgacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg
     2341 ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc
     2401 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2461 cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2521 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacagtgag ctgcagaaca
     2581 agctggaaga gttggatctg gactcgtaca agtaaagcgg ccgcgactct agatcataat
     2641 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     2701 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     2761 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2821 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     2881 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     2941 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3001 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3061 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3121 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3181 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3241 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3301 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3361 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3421 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     3481 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3541 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3601 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3661 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3721 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     3781 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     3841 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3901 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3961 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     4021 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     4081 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     4141 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     4201 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4261 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4321 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     4381 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4441 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4501 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4561 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4621 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4681 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4741 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4801 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     4861 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     4921 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     4981 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     5041 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     5101 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5161 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5221 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5281 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5341 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     5401 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5461 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5521 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5581 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5641 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5701 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5761 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5821 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5881 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5941 cgccatgcat
//
