LOCUS       sequence-368024-        6240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses miRFP2 and HO1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6240)
  AUTHORS   Babakhanova S, Jung EE, Namikawa K, Zhang H, Wang Y, Subach OM, 
            Korzhenevskiy DA, Rakitina TV, Xiao X, Wang W, Shi J, Drobizhev M, 
            Park D, Eisenhard L, Tang H, Koster RW, Subach FV, Boyden ES, 
            Piatkevich KD
  TITLE     Rapid directed molecular evolution of fluorescent proteins in 
            mammalian cells.
  JOURNAL   Protein Sci. 2022 Mar;31(3):728-751. doi: 10.1002/pro.4261. Epub 
            2021 Dec 30.
  PUBMED    34913537
REFERENCE   2  (bases 1 to 6240)
  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"; doi: "10.1002/pro.4261"; 
            journalName: "Protein Sci"; date: "2022-03"; volume: "31"; issue: 
            "3"; pages: "728-751"
COMMENT     Sequence Label: pCMV-miRFP2-IRES-HO1-N1
FEATURES             Location/Qualifiers
     source          1..6240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        175..478
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        479..682
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     633..653
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      716..725
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(1673..2247,2248..2250,2251..2259)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1840..1857)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2067..2086
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2258..2267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3126..3247
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3163..3182)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3217..3236
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3270..3725)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3407..3428)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3619..3638
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3752..3856
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3858..4215
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3858..4049
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3881..3901)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4066..4201
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4250..5044
                     /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             4250..5044
                     /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(4304..4323)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4914..4933
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5232..5251)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5276..5323
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5652..6240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6141..6160
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
      121 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      181 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      241 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      301 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      361 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      421 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      481 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      541 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      601 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      661 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctaga agcttgccac
      721 catggtcgca ggacacgcaa gtggaagccc cgactttgga accgccagtc cgagcgactg
      781 tgagcacgaa gaaatccatc tggccggatc catccagcca cacggaacac tgctggtggt
      841 ctccgagcct gaccatagaa taattcaggc atctgccaac gccgctgaat tcctgaatct
      901 gggaagtgtg ctgggaatcc ccctggctga gatcgacggg gatctactga tcaagattct
      961 gccccacctg gaccttaccg cagaaggaat gccagtggcc gtcaggtgcc gcattggaaa
     1021 cccctccaca gagtacgacg gactgatgca tcgaccacct gaaggaggac tgatcattga
     1081 gctggaacgg gctgggccat ccattgatct gagcggaaca ctggctcccg cactggagcg
     1141 aattcgaact gcaggatccc tgagggctct gtgcgacgat actgtactgc tgttccagca
     1201 gtgtaccggc tacgaccgag tgatggtcta tcggttcgat gaacgggggc acggagaggt
     1261 gtattcagaa attcatgtga ctggactgga gagctacttt ggcaacaggt atcctagctc
     1321 cctcgtgcca cagatggccc ggcggctgta cgtgagacag agggtgcgcg tcctggtaga
     1381 tgtcacctat cagcctgtgc cactggagcc aagactgagt cccctgactg gcagggacct
     1441 ggatatgtca gggtgttttc tgcgctctat gagtcctact cacctgcagt tcctgaagaa
     1501 catgggcgtg cgggcaacac tggtggtcag cctggtggtc ggaggcaaac tgtgggggct
     1561 ggtgatctgc caccattatc tgccacgctt catccatttt gagctgcgag caatttgtgt
     1621 gctgctggcc gaagctatcg caacccgcat tacagccctg taagcggccg cgcccctctc
     1681 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     1741 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     1801 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     1861 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     1921 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     1981 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     2041 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     2101 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
     2161 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     2221 ttttcctttg aaaaacacga tgataatatg gccacaacca tggcggctag cgccaccatg
     2281 agtgtcaact tagcttccca gttgcgggaa gggacgaaaa aatcccactc catggcggag
     2341 aacgtcggct ttgtcaaatg cttcctcaag ggcgttgtcg agaaaaattc ctaccgtaag
     2401 ctggttggca atctctactt tgtctacagt gccatggaag aggaaatggc aaaatttaag
     2461 gaccatccca tcctcagcca catttacttc cccgaactca accgcaaaca aagcctagag
     2521 caagacctgc aattctatta cggctccaac tggcggcaag aagtgaaaat ttctgccgct
     2581 ggccaagcct atgtggaccg agtccggcaa gtggccgcta cggcccctga attgttggtg
     2641 gcccattcct acacccgtta cctgggggat ctttccggcg gtcaaattct caagaaaatt
     2701 gcccaaaatg ccatgaatct ccacgatggt ggcacagctt tctatgaatt tgccgacatt
     2761 gatgacgaaa aggcttttaa aaatacctac cgtcaagcta tgaatgatct gcccattgac
     2821 caagccaccg ccgaacggat tgtggatgaa gccaatgacg cctttgccat gaacatgaaa
     2881 atgttcaacg aacttgaagg caacctgatc aaggcgatcg gcattatggt gttcaacagc
     2941 ctcacccgtc gccgcagtca aggcagcacc gaagttggcc tcgccacctc cgaaggctaa
     3001 gaattcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     3061 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     3121 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3181 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3241 tatcttaagg cgtaaattgt ttaaggcgta aattgtaagc gttaatattt tgttaaaatt
     3301 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     3361 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     3421 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     3481 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3541 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3601 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3661 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3721 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3781 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3841 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     3901 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3961 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4021 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4081 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4141 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4201 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4261 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4321 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4381 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     4441 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4501 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4561 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4621 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4681 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4741 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     4801 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4861 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4921 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4981 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     5041 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     5101 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5161 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg
     5221 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     5281 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg
     5341 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc
     5401 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
     5461 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
     5521 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5581 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5641 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5701 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5761 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5821 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5881 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5941 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6001 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6061 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6121 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6181 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
//
