LOCUS       sequence-368025-        5671 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 5671)
  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 5671)
  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: pmiRFP2-P2A-HO1-N1
FEATURES             Location/Qualifiers
     source          1..5671
                     /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"
     regulatory      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1557..1613
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     polyA_signal    2459..2580
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2496..2515)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2550..2569
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2587..3042)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2724..2745)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2936..2955
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3069..3173
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3175..3532
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3175..3366
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3198..3218)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3383..3518
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3567..4361
                     /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             3567..4361
                     /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(3621..3640)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4231..4250
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4549..4568)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4593..4640
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4969..5557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5458..5477
                     /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 gctagcgcca
      601 ccatggtcgc aggacacgca agtggaagcc ccgactttgg aaccgccagt ccgagcgact
      661 gtgagcacga agaaatccat ctggccggat ccatccagcc acacggaaca ctgctggtgg
      721 tctccgagcc tgaccataga ataattcagg catctgccaa cgccgctgaa ttcctgaatc
      781 tgggaagtgt gctgggaatc cccctggctg agatcgacgg ggatctactg atcaagattc
      841 tgccccacct ggaccttacc gcagaaggaa tgccagtggc cgtcaggtgc cgcattggaa
      901 acccctccac agagtacgac ggactgatgc atcgaccacc tgaaggagga ctgatcattg
      961 agctggaacg ggctgggcca tccattgatc tgagcggaac actggctccc gcactggagc
     1021 gaattcgaac tgcaggatcc ctgagggctc tgtgcgacga tactgtactg ctgttccagc
     1081 agtgtaccgg ctacgaccga gtgatggtct atcggttcga tgaacggggg cacggagagg
     1141 tgtattcaga aattcatgtg actggactgg agagctactt tggcaacagg tatcctagct
     1201 ccctcgtgcc acagatggcc cggcggctgt acgtgagaca gagggtgcgc gtcctggtag
     1261 atgtcaccta tcagcctgtg ccactggagc caagactgag tcccctgact ggcagggacc
     1321 tggatatgtc agggtgtttt ctgcgctcta tgagtcctac tcacctgcag ttcctgaaga
     1381 acatgggcgt gcgggcaaca ctggtggtca gcctggtggt cggaggcaaa ctgtgggggc
     1441 tggtgatctg ccaccattat ctgccacgct tcatccattt tgagctgcga gcaatttgtg
     1501 tgctgctggc cgaagctatc gcaacccgca ttacagccct ggaattcgga tccggagcta
     1561 ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctatgagtg
     1621 tcaacttagc ttcccagttg cgggaaggga cgaaaaaatc ccactccatg gcggagaacg
     1681 tcggctttgt caaatgcttc ctcaagggcg ttgtcgagaa aaattcctac cgtaagctgg
     1741 ttggcaatct ctactttgtc tacagtgcca tggaagagga aatggcaaaa tttaaggacc
     1801 atcccatcct cagccacatt tacttccccg aactcaaccg caaacaaagc ctagagcaag
     1861 acctgcaatt ctattacggc tccaactggc ggcaagaagt gaaaatttct gccgctggcc
     1921 aagcctatgt ggaccgagtc cggcaagtgg ccgctacggc ccctgaattg ttggtggccc
     1981 attcctacac ccgttacctg ggggatcttt ccggcggtca aattctcaag aaaattgccc
     2041 aaaatgccat gaatctccac gatggtggca cagctttcta tgaatttgcc gacattgatg
     2101 acgaaaaggc ttttaaaaat acctaccgtc aagctatgaa tgatctgccc attgaccaag
     2161 ccaccgccga acggattgtg gatgaagcca atgacgcctt tgccatgaac atgaaaatgt
     2221 tcaacgaact tgaaggcaac ctgatcaagg cgatcggcat tatggtgttc aacagcctca
     2281 cccgtcgccg cagtcaaggc agcaccgaag ttggcctcgc cacctccgaa ggctaaagcg
     2341 gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     2401 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     2461 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2521 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2581 aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2641 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2701 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2761 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2821 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2881 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2941 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     3001 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     3061 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3121 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     3181 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3241 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3301 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3361 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3421 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     3481 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     3541 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3601 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3661 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3721 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     3781 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3841 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3901 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3961 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4021 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4081 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4141 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4201 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4261 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4321 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     4381 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4441 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4501 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     4561 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     4621 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     4681 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     4741 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     4801 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4861 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4921 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4981 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5041 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5101 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5161 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5221 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5281 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5341 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5401 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5461 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5521 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5581 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5641 ttctgtggat aaccgtatta ccgccatgca t
//
