LOCUS       sequence-389479-        6204 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses the fusion protein of Keratin-miRFP720 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6204)
  AUTHORS   Zhang H, Papadaki S, Sun X, Wang X, Drobizhev M, Yao L, Rehbock M, 
            Koster RW, Wu L, Namikawa K, Piatkevich KD
  TITLE     Quantitative assessment of near-infrared fluorescent proteins.
  JOURNAL   Nat Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z.
  PUBMED    37666982
REFERENCE   2  (bases 1 to 6204)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z."
COMMENT     Sequence Label: pKeratin-miRFP720-N1
FEATURES             Location/Qualifiers
     source          1..6204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        86..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1945..2895
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 720 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP720
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACKRVAERLATQIGVMEE"
     polyA_signal    3017..3138
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3054..3073)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3108..3127
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3145..3600)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3282..3303)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3494..3513
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3627..3731
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3733..4090
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3733..3924
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3756..3776)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3941..4076
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4125..4919
                     /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             4125..4919
                     /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(4179..4198)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4789..4808
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5107..5126)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5151..5198
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5527..6115
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6016..6035
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact tttcaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
      601 tgaaccgtca gatccgctag caccatgagc ttcaccactc gctccacctt ctccaccaac
      661 taccggtccc tgggctctgt ccaggcgccc agctacggcg cccggccggt cagcagcgcg
      721 gccagcgtct atgcaggcgc tgggggctct ggttcccgga tctccgtgtc ccgctccacc
      781 agcttcaggg gcggcatggg gtccgggggc ctggccaccg ggatagccgg gggtctggca
      841 ggaatgggag gcatccagaa cgagaaggag accatgcaaa gcctgaacga ccgcctggcc
      901 tcttacctgg acagagtgag gagcctggag accgagaacc ggaggctgga gagcaaaatc
      961 cgggagcact tggagaagaa gggaccccag gtcagagact ggagccatta cttcaagatc
     1021 atcgaggacc tgagggctca gatcttcgca aatactgtgg acaatgcccg catcgttctg
     1081 cagattgaca atgcccgtct tgctgctgat gactttagag tcaagtatga gacagagctg
     1141 gccatgcgcc agtctgtgga gaacgacatc catgggctcc gcaaggtcat tgatgacacc
     1201 aatatcacac gactgcagct ggagacagag atcgaggctc tcaaggagga gctgctcttc
     1261 atgaagaaga accacgaaga ggaagtaaaa ggcctacaag cccagattgc cagctctggg
     1321 ttgaccgtgg aggtagatgc ccccaaatct caggacctcg ccaagatcat ggcagacatc
     1381 cgggcccaat atgacgagct ggctcggaag aaccgagagg agctagacaa gtactggtct
     1441 cagcagattg aggagagcac cacagtgctc accacacagt ctgctgaggt tggagctgct
     1501 gagacgacgc tcacagagct gagacgtaca gtccagtcct tggagatcga cctggactcc
     1561 atgagaaatc tgaaggccag cttggagaac agcctgaggg aggtggaggc ccgctacgcc
     1621 ctacagatgg agcagctcaa cgggatcctg ctgcaccttg agtcagagct ggcacagacc
     1681 cgggcagagg gacagcgcca ggcccaggag tatgaggccc tgctgaacat caaggtcaag
     1741 ctggaggctg agatcgccac ctaccgccgc ctgctggaag atggcgagga ctttaatctt
     1801 ggtgatgcct tggacagcag caactccatg caaaccatcc aaaagaccac cacccgccgg
     1861 atagtggatg gcaaagtggt gtctgagacc aatgacacca aagttctgag gcatcgaatt
     1921 ctgcagtcga cggtaccacc ggtcatggcg gaaggatccg tcgccaggca gcctgacctc
     1981 ttgacctgcg acgatgagcc gatccatatc cccggtgcca tccaaccgca tggactgctg
     2041 ctcgccctcg ccgccgacat gacgatcgtt gccggcagcg acaaccttcc cgaactcacc
     2101 ggactggcga tcggcgccct gatcggccgc tctgcggccg atgtcttcga ctcggagacg
     2161 cacaaccgtc tgacgatcgc cttggccgag cccggggcgg ccgtcggagc accgatcact
     2221 gtcggcttca cgatgcgaaa ggacgcaggc ttcatcggct cctggcatcg ccatgatcag
     2281 ctcatcttcc tcgagctcga gcctccccag cgggacgtcg ccgagccgca ggcgttcttc
     2341 cgccgcacca acagcgccat ccgccgcctg caggccgccg aaaccttgga aagcgcctgc
     2401 gccgccgcgg cgcaagaggt gcggaagatt accggcttcg atcgggtgat gatctatcgc
     2461 ttcgcctccg acttcagcgg gtccgtgatc gcagaggatc ggtgcgccga ggtcgagtca
     2521 aaactaggcc tgcactatcc tgcctcattc atcccggcgc aggcccgtcg gctctatacc
     2581 atcaacccgg tacggatcat tcccgatatc aattatcggc cggtgccggt caccccagac
     2641 ctcaatccgg tcaccgggcg gccgattgat cttagcttcg ccatcctgcg cagcgtctcg
     2701 cccaaccatc tggagttcat gcgcaacata ggcatgcacg gcacgatgtc gatctcgatt
     2761 ttgcgcggcg agcgactgtg gggattgatc gtttgccatc accgaacgcc gtactacgtc
     2821 gatctcgatg gccgccaagc ctgcaagaga gtcgccgaga gactggccac ccagatcggc
     2881 gtgatggaag agtaggcggc cgcgactcta gatcataatc agccatacca catttgtaga
     2941 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     3001 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     3061 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3121 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     3181 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3241 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3301 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3361 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3421 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3481 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3541 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3601 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3661 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3721 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     3781 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3841 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3901 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3961 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4021 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4081 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4141 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4201 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4261 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     4321 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4381 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4441 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4501 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4561 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4621 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     4681 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4741 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4801 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4861 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4921 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4981 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5041 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     5101 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     5161 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     5221 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     5281 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     5341 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     5401 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5461 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5521 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5581 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5641 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5701 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5761 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5821 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5881 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5941 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6001 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6061 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6121 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6181 tcctgcgtta tcccctgatt ctgt
//
