LOCUS       sequence-363931-        7056 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coexpression of PhiC31o recombinase and EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7056)
  AUTHORS   Kozhukhar N, Spadafora D, Rodriguez YAR, Alexeyev MF
  TITLE     A Method for In Situ Reverse Genetic Analysis of Proteins Involved 
            mtDNA Replication.
  JOURNAL   Cells. 2022 Jul 11;11(14). pii: cells11142168. doi: 
            10.3390/cells11142168.
  PUBMED    35883613
REFERENCE   2  (bases 1 to 7056)
  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: "Cells."; 
            date: "2022-07-11"; volume: "11(14). pii: cells11142168. doi"; 
            pages: " 10.3390/cells11142168"
COMMENT     Sequence Label: pMA4854
FEATURES             Location/Qualifiers
     source          1..7056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    26..147
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(148..861,862..864,865..867))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(151..861,862..864,865..867))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(193..214)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     542..561
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      864..873
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(883..1144)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     enhancer        1201..1504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1505..1708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1659..1679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1797..1806
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1803..3617
                     /codon_start=1
                     /product="integrase from phage phi-C31"
                     /label=phage phi-C31 integrase
                     /translation="MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDG
                     GRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIP
                     IVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQREL
                     GGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWRE
                     IKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRIL
                     RDPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQA
                     WLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPG
                     QHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERA
                     NLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAE
                     APKLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPI
                     EKRASITWAKPPTDDDEDDAQDGTEDVAA"
     CDS             3618..3638
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3840..3961
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(3972..4427)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4109..4130)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4321..4340
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4454..4558
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4560..4917
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4560..4751
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4583..4603)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4768..4903
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4952..5746
                     /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             4952..5746
                     /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(5006..5025)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5616..5635
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5934..5953)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5978..6025
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6354..6942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6843..6862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattag ggtatacaga catgataaga tacattgatg agtttggaca aaccacaact
       61 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
      121 accattataa gctgcaataa acaagtttta cttgtacagc tcgtccatgc cgagagtgat
      181 cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt
      241 gctcagggcg gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc
      301 gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg
      361 gcggatcttg aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt
      421 gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc
      481 gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg
      541 ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg
      601 catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg
      661 cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt
      721 gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac
      781 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
      841 gaacagctcc tcgcccttgc tcaccatggt ggcgagcttg gaggtgcaca ccaatgtggt
      901 gaatggtcaa atggcgttta ttgtatcgag ctaggcactt aaatacaata tctctgcaat
      961 gcggaattca gtggttcgtc caatccatgt cagacccgtc tgttgccttc ctaataaggc
     1021 acgatcgtac caccttactt ccaccaatcg gcatgcacgg tgctttttct ctccttgtaa
     1081 ggcatgttgc taactcatcg ttaccatgtt gcaagactac aagagtattg cataagacta
     1141 cattccctaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     1201 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     1261 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     1321 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     1381 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     1441 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     1501 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     1561 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     1621 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     1681 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagatcga
     1741 attccatcaa gcttatcgat accgtcgacg gtatcgataa gcttgatgcg gaattcgcca
     1801 ccatggatac ctacgccgga gcctacgaca gacagagccg ggagagagag aacagcagcg
     1861 ccgccagccc cgccacccag agaagcgcca acgaggataa ggccgccgat ctgcagagag
     1921 aggtggagag ggacggcggc agattcagat ttgtgggcca cttcagcgag gcccctggca
     1981 ccagcgcctt cggcaccgcc gagagacccg agttcgagag aatcctgaac gagtgtaggg
     2041 ccggcaggct gaacatgatc atcgtgtacg acgtgtcccg gttcagcagg ctgaaggtga
     2101 tggacgccat ccctatcgtg tccgagctgc tggccctggg cgtgaccatc gtgtccaccc
     2161 aggaaggcgt ctttagacag ggcaacgtga tggacctgat ccacctgatc atgaggctgg
     2221 acgccagcca caaggagagc agcctgaaga gcgccaagat cctggacacc aagaacctgc
     2281 agagggagct gggcggctat gtgggcggca aggcccccta cggcttcgag ctggtgtccg
     2341 agaccaagga gatcacccgg aacggcagga tggtgaacgt ggtgatcaac aagctggccc
     2401 acagcaccac ccccctgacc ggccccttcg agtttgagcc cgacgtgatc aggtggtggt
     2461 ggcgggagat caagacccac aagcacctgc ctttcaagcc cggcagccag gccgccatcc
     2521 accccggcag catcaccggc ctgtgtaaga gaatggacgc cgacgccgtg cccaccagag
     2581 gcgagaccat cggcaagaaa accgccagca gcgcctggga ccccgccacc gtgatgagaa
     2641 tcctgaggga ccctaggatc gccggcttcg ccgccgaggt gatctacaag aagaagcccg
     2701 acggcacccc caccaccaag atcgagggct acagaatcca gagagacccc atcaccctga
     2761 gacctgtgga gctggactgt ggccctatca tcgagcctgc cgagtggtac gagctgcagg
     2821 cctggctgga cggcagaggc agaggcaagg gcctgagcag aggccaggcc atcctgagcg
     2881 ccatggacaa gctgtactgt gagtgtggcg ccgtgatgac cagcaagaga ggcgaggaga
     2941 gcatcaagga cagctaccgg tgccggagaa gaaaggtggt ggaccccagc gcccctggcc
     3001 agcacgaggg cacctgtaat gtgagcatgg ccgccctgga caagttcgtg gccgagcgga
     3061 tcttcaacaa gatccggcac gccgagggcg acgaggagac cctggccctg ctgtgggagg
     3121 ccgccagaag attcggcaag ctgaccgagg cccccgagaa gagcggcgag agggccaacc
     3181 tggtggccga gagagccgac gccctgaacg ccctggagga gctgtacgag gacagagccg
     3241 ccggagccta tgacggccct gtgggcagga agcacttcag aaagcagcag gccgccctga
     3301 ccctgagaca gcagggcgcc gaggaaagac tggccgagct ggaggccgcc gaggccccta
     3361 agctgcccct ggatcagtgg ttccccgagg atgccgacgc cgaccccacc ggccccaagt
     3421 cctggtgggg cagagccagc gtggacgaca agagggtgtt cgtgggcctg ttcgtggata
     3481 agatcgtggt gaccaagagc accaccggca ggggccaggg cacccccatc gagaagagag
     3541 ccagcatcac ctgggccaag cctcccaccg acgacgacga ggatgacgcc caggacggca
     3601 ccgaggacgt ggccgcccct aagaaaaagc ggaaagtgtg aaagcttctc gaggcatcga
     3661 attcctgcag cccgggggat cccccgggct gcaggaattc gatagcttga tgatccaccg
     3721 gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     3781 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     3841 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     3901 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     3961 aacgcgcgcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4021 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4081 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4141 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4201 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4261 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4321 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4381 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     4441 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4501 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     4561 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     4621 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     4681 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4741 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4801 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4861 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     4921 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4981 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     5041 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     5101 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     5161 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     5221 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     5281 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     5341 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     5401 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     5461 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     5521 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     5581 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     5641 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     5701 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     5761 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     5821 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5881 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     5941 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     6001 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     6061 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     6121 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     6181 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     6241 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6301 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6361 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6421 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6481 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6541 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6601 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6661 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6721 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6781 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6841 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6901 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6961 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     7021 cctgattctg tggataaccg tattaccgcc atgcat
//
