LOCUS       sequence-368010-        5661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses miRFP2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5661)
  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 5661)
  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: pAAV-CAG-miRFP2
FEATURES             Location/Qualifiers
     source          1..5661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        281..558
                     /label=chicken beta-actin promoter
     primer_bind     837..859
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     921..940
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      973..982
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1945..2533
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1998..2018)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2416..2427)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2576..2697
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2587..2606)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2641..2660
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2859..2988
                     /label=AAV2 ITR
     rep_origin      complement(3225..3813)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3305..3324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3984..4775,4776..4844))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4607..4626
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4845..4949)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4975..5430)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5112..5133)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5324..5343
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5502..5631
                     /label=AAV2 ITR
ORIGIN
        1 tatctacgta gccatgctct aggaagagta ccattgacgt caataatgac gtatgttccc
       61 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      121 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      181 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      241 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
      301 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
      361 taattatttt gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc gggggggggg
      421 ggggggggsg gggggsgggg sggggsgagg cggagaggtg cggcggcagc caatcagagc
      481 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      541 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
      601 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
      661 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct
      721 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
      781 ggctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
      841 cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt
      901 ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa
      961 agaattggat ccgccaccat ggtcgcagga cacgcaagtg gaagccccga ctttggaacc
     1021 gccagtccga gcgactgtga gcacgaagaa atccatctgg ccggatccat ccagccacac
     1081 ggaacactgc tggtggtctc cgagcctgac catagaataa ttcaggcatc tgccaacgcc
     1141 gctgaattcc tgaatctggg aagtgtgctg ggaatccccc tggctgagat cgacggggat
     1201 ctactgatca agattctgcc ccacctggac cttaccgcag aaggaatgcc agtggccgtc
     1261 aggtgccgca ttggaaaccc ctccacagag tacgacggac tgatgcatcg accacctgaa
     1321 ggaggactga tcattgagct ggaacgggct gggccatcca ttgatctgag cggaacactg
     1381 gctcccgcac tggagcgaat tcgaactgca ggatccctga gggctctgtg cgacgatact
     1441 gtactgctgt tccagcagtg taccggctac gaccgagtga tggtctatcg gttcgatgaa
     1501 cgggggcacg gagaggtgta ttcagaaatt catgtgactg gactggagag ctactttggc
     1561 aacaggtatc ctagctccct cgtgccacag atggcccggc ggctgtacgt gagacagagg
     1621 gtgcgcgtcc tggtagatgt cacctatcag cctgtgccac tggagccaag actgagtccc
     1681 ctgactggca gggacctgga tatgtcaggg tgttttctgc gctctatgag tcctactcac
     1741 ctgcagttcc tgaagaacat gggcgtgcgg gcaacactgg tggtcagcct ggtggtcgga
     1801 ggcaaactgt gggggctggt gatctgccac cattatctgc cacgcttcat ccattttgag
     1861 ctgcgagcaa tttgtgtgct gctggccgaa gctatcgcaa cccgcattac agccctgtaa
     1921 gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     1981 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     2041 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     2101 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     2161 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     2221 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     2281 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     2341 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     2401 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     2461 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     2521 gccgcctccc cgcatcgata ccgtcgaccc gggcggccgc ttcgagcaga catgataaga
     2581 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     2641 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     2701 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa
     2761 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atcttcctag agcatggcta
     2821 cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt
     2881 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     2941 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcattaatga
     3001 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3061 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3121 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3181 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3241 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3301 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3361 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3421 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3481 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3541 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3601 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3661 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3721 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3781 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3841 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3901 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3961 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4021 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4081 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4141 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4201 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4261 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4321 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4381 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4441 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4501 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4561 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4621 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4681 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4741 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4801 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4861 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4921 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     4981 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     5041 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     5101 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     5161 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     5221 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     5281 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     5341 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     5401 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
     5461 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctgcgcgct cgctcgctca
     5521 ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
     5581 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc ttgtagttaa
     5641 tgattaaccc gccatgctac t
//
