LOCUS       sequence-226732-        6036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PKAcs-GFP fusion in mammalian cells with flexible linker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6036)
  AUTHORS   Norris JL, Hughes RM
  TITLE     protaTETHER - a method for the incorporation of variable linkers in 
            protein fusions reveals impacts of linker flexibility in a PKAc-GFP 
            fusion protein.
  JOURNAL   FEBS Open Bio. 2018 Apr 25;8(6):1029-1042. doi: 
            10.1002/2211-5463.12414. eCollection 2018 Jun.
  PUBMED    29928581
REFERENCE   2  (bases 1 to 6036)
  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: 
            "10.1002/2211-5463.12414"; journalName: "FEBS Open Bio"; date: 
            "2018-04-25- 25"; volume: "8"; issue: "6"; pages: "1029-1042"
COMMENT     Sequence Label: PKAcs-GGS4X-GFP
FEATURES             Location/Qualifiers
     source          1..6036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..363
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        364..567
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     518..538
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     564..588
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          703..812
                     /label=CMV intron
                     /note="modified intron A from human cytomegalovirus (CMV)"
     primer_bind     758..777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        758..776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1964..1992)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2587..2607
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2824..2945
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2861..2880)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2915..2934
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2952..3407)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3089..3110)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3301..3320
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3434..3538
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3540..3897
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3540..3731
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3563..3583)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3748..3883
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3932..4726
                     /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             3932..4726
                     /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(3986..4005)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4596..4615
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4914..4933)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4958..5005
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5334..5922
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5823..5842
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctag tattagtcat cgctattacc
      361 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      421 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      481 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      541 cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc
      601 catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct ccgcggccgg
      661 gaacggtgca ttggaacgcg gattccccgt gccaagagtg acgtaagtac cgcctataga
      721 ctctataggc acaccccttt ggctcttatg catgaattaa tacgactcac tatagggaga
      781 cagactgttc ctttcctggg tcttttctgc aggcaccgtc gtcgacttaa cagatctcga
      841 gatgggcaac gccgccgccg ccaagaaggg cagcgagcag gagagcgtga aagagttcct
      901 agccaaagcc aaggaagatt tcctgaaaaa atgggagacc ccttctcaga atacagccca
      961 gttggatcag tttgatagaa tcaagaccct tggcaccggc tcctttgggc gagtgatgct
     1021 ggtgaagcac aaggagagtg ggaaccacta cgccatgaag atcttagaca agcagaaggt
     1081 ggtgaagcta aagcagatcg agcagactct gaatgagaag cgcatcctgc aggccgtcaa
     1141 cttcccgttc ctggtcaaac ttgaattctc cttcaaggac aactcaaacc tgtacatggt
     1201 catggagtat gtagctggtg gcgagatgtt ctcccaccta cggcggattg gaaggttcag
     1261 cgagccccat gcccgtttct acgcggcgca gatcgtcctg acctttgagt atctgcactc
     1321 cctggacctc atctaccggg acctgaagcc cgagaatctt ctcatcgacc agcagggcta
     1381 tattcaggtg acagacttcg gttttgccaa gcgtgtgaaa ggccgtactc ggaccttgtg
     1441 tgggacccct gagtacttgg cccccgagat tatcctgagc aaaggctaca acaaggctgt
     1501 ggactggtgg gctctcggag tcctcatcta cgagatggct gctggttacc cacccttctt
     1561 cgctgaccag cctatccaga tctatgagaa aatcgtctct gggaaggtgc ggttcccatc
     1621 ccacttcagc tctgacttga aggacctgct gcggaacctt ctgcaagtgg atctcaccaa
     1681 gcgctttggg aacctcaaga acggggtcaa tgacatcaag aaccacaagt ggtttgccac
     1741 gactgactgg attgccatct atcagagaaa ggtggaagct cccttcatac caaagtttaa
     1801 aggccctggg gacacgagta actttgacga ctatgaggag gaagagatcc gggtctccat
     1861 caatgagaag tgtggcaagg agtttactga gtttggcggc agcggcggca gcggcggcag
     1921 cggcggcagc atggctagca aaggagaaga actcttcact ggagttgtcc caattcttgt
     1981 tgaattagat ggtgatgtta acggccacaa gttctctgtc agtggagagg gtgaaggtga
     2041 tgcaacatac ggaaaactta ccctgaagtt catctgcact actggcaaac tgcctgttcc
     2101 atggccaaca ctagtcacta ctctgtgcta tggtgttcaa tgcttttcaa gatacccgga
     2161 tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag
     2221 gaccatcttc ttcaaagatg acggcaacta caagacacgt gctgaagtca agtttgaagg
     2281 tgataccctt gttaatagaa tcgagttaaa aggtattgac ttcaaggaag atggcaacat
     2341 tctgggacac aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa
     2401 acaaaagaat ggaatcaaag tgaacttcaa gacccgccac aacattgaag atggaagcgt
     2461 tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc
     2521 agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga
     2581 ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca ggatgaactg
     2641 tacaactgag aattctgcag tcgacggtac cgcgggcccg ggatccaccg ggtacaagta
     2701 aagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     2761 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     2821 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2881 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2941 tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3001 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3061 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     3121 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     3181 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     3241 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     3301 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     3361 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     3421 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3481 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     3541 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3601 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3661 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3721 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3781 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     3841 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     3901 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     3961 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4021 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4081 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     4141 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4201 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4261 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4321 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4381 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4441 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4501 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4561 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4621 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4681 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4741 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4801 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4861 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     4921 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     4981 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     5041 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     5101 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     5161 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     5221 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5281 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5341 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5401 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5461 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5521 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5581 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5641 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5701 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5761 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5821 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5881 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5941 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6001 cctgattctg tggataaccg tattaccgcc atgcat
//
