LOCUS       sequence-368300-        8222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses miRFP2 and HO1in worm cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8222)
  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 8222)
  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: pSF11-wmiRFP2-T2A-HO1
FEATURES             Location/Qualifiers
     source          1..8222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3900..3953
                     /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"
     primer_bind     complement(5792..5814)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5914..5933
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6015..6033)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6101..6205
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6206..6274,6275..7066)
                     /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     complement(6424..6443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7237..7825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7726..7745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7979..7996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8113..8134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8149..8154,8155..8172,8173..8179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8187..8203
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     8192..8214
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(8211..8222,1..5)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(8211..8222,1..5)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgaccatga ttacgccaag cttgcatgcg cggccgcact gactgggccg gccggatcca
       61 taggtatgcc tgtcattctc tatttttaaa cgttcctgca gcagtagcac aacagagcca
      121 gcagttttcc tcttcttctc catcttcttc tgcttcttcc tctcttccca aaaaaaaaaa
      181 aaaaaaaaaa nngtgagggg gctcactgcc ccccccccnn tccccccccc ctcttctcct
      241 tgaagctcat ccagacgtcg ctgtcctcct cctcctcgtc cccggggcgc tgtgtgtgtg
      301 tgtgtgtgct ccgtgagcat gtagcagctt cagcccattt aaaggcgcac actctctgca
      361 tctctgcgtg tctcgacact cgttttttct cttcttttct gctgcacaca cacacacaca
      421 cacacacacg ctcatgccag agcacttctt gctccggggg gctccgcgcg gagcccgaga
      481 ggacctcctc ctcctttttc ttcttcgctt tttagttctt cttgcctcgg tctcgcccct
      541 tgttttgctc tccatgtagt atatacatct gggtgtgtgt gtgtgtgtgc acctttggag
      601 cattttttga acctttccag aactttggag caacattttc agcttgttca gtgcactttt
      661 taggctgaaa atcctatttt taggttttcg aaagtttttt tggagcattt ttcgtgcaat
      721 ttaggtgcaa aattaaaaaa aagggttttg tagtgaatta aaattttttt gaaactttcg
      781 cctttttaaa aaaatgtttc taatatcttt aaaaattgga atattttgga aaacaatttg
      841 ttttcaattg gcaatttatt aattctgagc ttctgagcgt aaattttttt ttattttgga
      901 gcaattctga gatgtcttag tgcatttttc gggcatacat aaaaaaaaat ttatcttaat
      961 aataaaaacc ttaaatttag gcttcaccgt ctggaaattc aattttttca gctgaaaaac
     1021 tttcaaaaaa aaaaaaatta aattttcaca tttattttct ctggaatttt ggagcaattt
     1081 caaagctgtc ttagtgcatt tttagtacac aaatattttt tcatttttct taaattttaa
     1141 taatctaagc ttaaataaag gcttcaccgt ctgaaaatgc aacttttttc agatgaaata
     1201 ttaaaaaaaa aacctctttt attaattttg tgctccaaac tttcccacgg tacgcaaatc
     1261 atgtaatatg tacttttaaa cattattttt ccaatcaaaa atatccaaca gcttttcctt
     1321 acaatttgaa aatttttatt aaaatttcaa aaaaattttt ttttttttta atttcgaatt
     1381 tttttaaact tgttgtaact tttttccctg tcggcgccaa ttaacaaaat acgtagaatt
     1441 ttgcgcttat tgttttgtta gttaacaaat ttttgacaac tttcttatca gctgagattt
     1501 aggtgtttca gttcgcaatc tgcgtctctc cttgctctcc tctattttaa aacccccata
     1561 actttgttct tgtgagagtt atcaaaaaat tgtcaactaa caaaatatag agcacaaaaa
     1621 tatgcacatt ttgtatgttg acaatttttt gatacctttt ttagttttcg agatatctta
     1681 gttttagttt agctgtacct ttttctggca aaaaacttcc aaatttcggc aatttttcgg
     1741 caaatttccc ggcaaatttc gccaagtaca tcgggctctc agacaagtgg tctgtgagtg
     1801 gaagtcgata aaagtagttt acgtgctctc cgtccccccg cttcttctcc gtctctgctt
     1861 ctatttctcc attgactagt cccactactt tttacggggc ggagggctct cttgcactcc
     1921 agcttcttct ctttttgtgc tcttttttgc ccgtatgtgg gtgctcattt atttttcttt
     1981 ggttttaaaa taaattcggt ttattaggct actcgtaaaa atgaagaaaa aacttcaaat
     2041 tcaaacgaca ttaaaaaaaa atttaataaa aattttttga attgctcaaa agccccacct
     2101 atatatatat atagctccgc ctacttctcg cacagacccc gcccactttt tgactccgcc
     2161 cccttattgg caaaagttct tgaaaaaaat aaaaataaaa attcaaattg aaacaatttt
     2221 tttttaatga agtagttttt ttctaaattg atgaaaattt ctaaacagtt tacaagcacc
     2281 gcccactttt tgactccgcc cctttgttgg aaaaattctt gtaaaaaaaa ataaaaaata
     2341 aaagcttctc tttcaaacga tttttagttg gtctttgtat gaaaagtttt tttcctaaaa
     2401 tagaaatgtc tgaataattt gcaagctccg cccttttctt acccagactc cgccccttta
     2461 ttgactaatt tttttttgtc catttacctg aagttcaaaa aaaaaattta cagtttcata
     2521 gagtaataat ttttcaaaaa tctctttttc caatgtttaa tgtgtcatta taaacccgcc
     2581 cagttttaag ctccgcccat ttaggccccg cccatttatg acccaaatat ttttttctct
     2641 attcacccgt ggatttccct ccaacaagtt accccaaaaa taaaaataaa aattcctgca
     2701 ggcgcccacc gggatccggc gcgcctctag aggatccccg ggattggcca aaggacccaa
     2761 aggtatgttt cgaatgatac taacataaca tagaacattt tcaggaggac ccttggctag
     2821 cgtcgacggt accaaaaatg gtcgctggac acgcttctgg atctccagat ttcggaaccg
     2881 cttctccatc tgattgcgaa cacgaggaga tccaccttgc tggatctatc caaccacatg
     2941 gaacccttct tgttgtttct gagccagatc accgcatcat ccaagcttcc gctaacgctg
     3001 ccgagttcct taaccttgga tccgttcttg gaatcccact tgccgagatc gacggagacc
     3061 ttcttatcaa ggtaagttta aacatatata tactaactaa ccctgattat ttaaattttc
     3121 agatccttcc acaccttgac ctcaccgctg agggaatgcc agttgctgtt cgctgccgta
     3181 tcggaaaccc atctactgag tacgatggac ttatgcaccg cccaccagag ggaggactta
     3241 tcatcgagct tgagcgtgcc ggaccatcta tcgacctttc cggaaccctt gccccagctc
     3301 ttgagcgcat ccgtaccgct ggatcccttc gtgccctttg cgacgacacc gtccttcttt
     3361 tccaacaatg cactggatac gatcgtgtta tggtttaccg cttcgacgag cgcggacacg
     3421 gagaggttta ctctgagatc cacgttaccg gacttgagtc ttacttcgga aatcgctacc
     3481 catcctccct tgttccacaa atggctcgtc gtctttacgt tcgtcaacgt gttcgtgtcc
     3541 ttgttgacgt tacctaccaa ccagttccac ttgagccacg tctttcccca cttaccggac
     3601 gtgatcttga catgtctgga tgcttccttc gttccatgtc tccaacccac cttcaattcc
     3661 ttaaggtaag tttaaacagt tcggtactaa ctaaccatac atatttaaat tttcagaaca
     3721 tgggagttcg tgctaccctt gttgtttccc ttgttgttgg aggaaagctt tggggacttg
     3781 ttatctgcca tcactacctt ccacgtttca tccacttcga acttcgtgcc atctgcgtcc
     3841 ttcttgctga ggccatcgct acccgtatca ccgctctcgg atacgacatc ggatccggag
     3901 agggacgcgg atcccttctt acctgcggag atgttgagga aaacccagga ccaatggagc
     3961 gcccacaacc agattctatg ccacaagacc tttccgaggc ccttaaggag gctaccaagg
     4021 aggttcacac ccaagctgag aacgctgagt tcatgcgcaa cttccaaaag ggacaagtta
     4081 cccgtgatgg attcaagctt gtcatggctt ccctctacca catctacgtc gctcttgagg
     4141 aggagatcga gcgcaacaag gtaagtttaa acatatatat actaactaac cctgattatt
     4201 taaattttca ggagtctcca gttttcgctc cagtttactt cccagaagag ctccaccgta
     4261 aggctgccct tgagcaagac cttgccttct ggtatggacc acgctggcaa gaagttatcc
     4321 catacactcc agctatgcaa cgctacgtta agcgtcttca cgaggtcgga cgtaccgagc
     4381 cagagcttct tgtcgctcac gcttacaccc gttaccttgg agacctttcc ggaggacaag
     4441 tccttaaggt aagtttaaac agttcggtac taactaacca tacatattta aattttcaga
     4501 agatcgctca aaaggctctt gaccttccat cttccggaga gggacttgcc ttcttcactt
     4561 tcccaaacat cgcttctgcc actaagttca agcaacttta ccgctctcgt atgaactctc
     4621 ttgagatgac cccagctgtc cgccaacgtg tcatcgagga ggctaaggta agtttaaaca
     4681 tgattttact aactaactaa tctgatttaa attttcagac cgctttcctt cttaacatcc
     4741 aacttttcga agagcttcaa gagcttctta cccacgacac caaggaccaa tctccatccc
     4801 gtgccccagg acttcgtcaa cgtgcctcca acaaggtcca agattccgct ccagttgaga
     4861 ctccacgtgg aaagccacca cttaacaccc gttcccaagc cccacttctt cgttgggtcc
     4921 ttaccctctc cttccttgtc gccaccgtcg ctgtcggact ttacgccatg taagagctcc
     4981 gcatcggccg ctgtcatcag atcgccatct cgcgcccgtg cctctgactt ctaagtccaa
     5041 ttactcttca acatccctac atgctctttc tccctgtgct cccaccccct atttttgtta
     5101 ttatcaaaaa acttctctta atttctttgt tttttagctt cttttaagtc acctctaaca
     5161 atgaaattgt gtagattcaa aaatagaatt aattcgtaat aaaaagtcga aaaaaattgt
     5221 gctccctccc cccattaata ataattctat cccaaaatct acacaatgtt ctgtgtacac
     5281 ttcttatgtt ttttacttct gataaatttt tttgaaacat catagaaaaa accgcacaca
     5341 aaatacctta tcatatgtta cgtttcagtt tatgaccgca atttttattt cttcgcacgt
     5401 ctgggcctct catgacgtca aatcatgctc atcgtgaaaa agttttggag tatttttgga
     5461 atttttcaat caagtgaaag tttatgaaat taattttcct gcttttgctt tttggggttt
     5521 cccctattgt ttgtcaagat ttcgaggacg gcgtttttct tgctaaaatc acaagtattg
     5581 atgagcacga tgcaagaaag atcggaagaa ggtttgggtt tgaggctcag tggaaggtga
     5641 gtagaagttg ataatttgaa agtggagtag tgtctatggg gtttttgcct taaatgacag
     5701 aatacattcc caatatacca aacataactg tttctactag tcggccgtac gggccctttc
     5761 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     5821 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     5881 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     5941 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     6001 ggccttaatt aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     6061 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     6121 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     6181 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     6241 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     6301 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     6361 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     6421 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     6481 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6541 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6601 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6661 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6721 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6781 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6841 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6901 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6961 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     7021 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     7081 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     7141 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     7201 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     7261 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     7321 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     7381 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     7441 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7501 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7561 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7621 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7681 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7741 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7801 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7861 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7921 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7981 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     8041 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     8101 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     8161 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     8221 ct
//
