LOCUS       sequence-226741-        6060 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 6060)
  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 6060)
  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-GGSD5X-GFP
FEATURES             Location/Qualifiers
     source          1..6060
                     /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(1988..2016)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2611..2631
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2848..2969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2885..2904)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2939..2958
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2976..3431)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3113..3134)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3325..3344
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3458..3562
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3564..3921
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3564..3755
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3587..3607)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3772..3907
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3956..4750
                     /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             3956..4750
                     /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(4010..4029)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4620..4639
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4938..4957)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4982..5029
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5358..5946
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5847..5866
                     /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 agcgacggcg gcagcgacgg
     1921 cggcagcgac ggcggcagcg acggcggcag cgacatggct agcaaaggag aagaactctt
     1981 cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaacggcc acaagttctc
     2041 tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctga agttcatctg
     2101 cactactggc aaactgcctg ttccatggcc aacactagtc actactctgt gctatggtgt
     2161 tcaatgcttt tcaagatacc cggatcatat gaaacggcat gactttttca agagtgccat
     2221 gcccgaaggt tatgtacagg aaaggaccat cttcttcaaa gatgacggca actacaagac
     2281 acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt taaaaggtat
     2341 tgacttcaag gaagatggca acattctggg acacaaattg gaatacaact ataactcaca
     2401 caatgtatac atcatggcag acaaacaaaa gaatggaatc aaagtgaact tcaagacccg
     2461 ccacaacatt gaagatggaa gcgttcaact agcagaccat tatcaacaaa atactccaat
     2521 tggcgatggc cctgtccttt taccagacaa ccattacctg tccacacaat ctgccctttc
     2581 gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa cagctgctgg
     2641 gattacacat ggcaggatga actgtacaac tgagaattct gcagtcgacg gtaccgcggg
     2701 cccgggatcc accgggtaca agtaaagcgg ccgcgactct agatcataat cagccatacc
     2761 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2821 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2881 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2941 ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt
     3001 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3061 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     3121 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3181 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3241 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3301 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3361 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3421 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3481 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3541 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3601 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3661 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3721 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3781 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3841 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3901 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     3961 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4021 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4081 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4141 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4201 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4261 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4321 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4381 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4441 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4501 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4561 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4621 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4681 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4741 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4801 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4861 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4921 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     4981 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     5041 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5101 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5161 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5221 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5281 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5341 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5401 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5461 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5521 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5581 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5641 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5701 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5761 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5821 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5881 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5941 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6001 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
