LOCUS       sequence-393633-        4602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of CFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4602)
  AUTHORS   Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur ELF
  TITLE     Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and 
            ARF6 GTPase disrupts the axonal transport of autophagosomes.
  JOURNAL   Cell Rep. 2023 May 1;42(5):112448. doi: 
            10.1016/j.celrep.2023.112448.
  PUBMED    37133994
REFERENCE   2  (bases 1 to 4602)
  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"; journalName: "Cell Rep.";
            date: "2023-05-1"; volume: "42(5):112448. doi"; pages: " 
            10.1016/j.celrep.2023.112448"
COMMENT     Sequence Label: PGK CFP
FEATURES             Location/Qualifiers
     source          1..4602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        130..640
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     559..577
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      656..665
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(662..664,665..667,668..1381)
                     /codon_start=1
                     /product="enhanced CFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(707..728)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(968..987)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1315..1336
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1504..1625
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1541..1560)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1595..1614
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1632..2087)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1769..1790)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1981..2000
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2114..2218
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2220..2577
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2220..2411
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2243..2263)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2428..2563
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2612..3406
                     /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             2612..3406
                     /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(2666..2685)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3276..3295
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3594..3613)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3638..3685
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4014..4602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4503..4522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
      121 ttaatccacg gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg
      181 cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt
      241 cttcacgtcc gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc
      301 gacgcttcct gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac
      361 gtgacaaacg gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc
      421 aatggcagcg cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc
      481 cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc
      541 cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt
      601 cggctccctc gttgaccgaa tcaccgacct ctctccccag gtggatccac cggtcgccac
      661 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      721 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      781 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      841 cctcgtgacc accctgacct ggggcgtgca gtgcttcagc cgctaccccg accacatgaa
      901 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
      961 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1021 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1081 caagctggag tacaactaca tcagccacaa cgtctatatc accgccgaca agcagaagaa
     1141 cggcatcaag gccaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1201 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1261 ctacctgagc acccagtcca aactgagcaa agaccccaac gagaagcgcg atcacatggt
     1321 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     1381 aagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     1441 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     1501 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1561 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     1621 tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1681 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     1741 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     1801 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     1861 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     1921 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     1981 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2041 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     2101 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2161 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     2221 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2281 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2341 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2401 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2461 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     2521 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     2581 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     2641 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     2701 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     2761 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     2821 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     2881 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     2941 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3001 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     3061 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     3121 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3181 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     3241 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     3301 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     3361 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3421 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     3481 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3541 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     3601 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     3661 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     3721 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     3781 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     3841 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     3901 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3961 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4021 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4081 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4141 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4201 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4261 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4321 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4381 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4441 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4501 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4561 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aa
//
