LOCUS       sequence-226724-        6033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PKAcs-GFP fusion in mammalian cells with structured 
            linker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6033)
  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 6033)
  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-L10X-GFP
FEATURES             Location/Qualifiers
     source          1..6033
                     /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(1961..1989)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2584..2604
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2821..2942
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2858..2877)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2912..2931
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2949..3404)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3086..3107)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3298..3317
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3431..3535
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3537..3894
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3537..3728
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3560..3580)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3745..3880
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3929..4723
                     /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             3929..4723
                     /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(3983..4002)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4593..4612
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4911..4930)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4955..5002
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5331..5919
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5820..5839
                     /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 gtttcttctt cttcttcttc ttcttcttct
     1921 tcttcttatg gctagcaaag gagaagaact cttcactgga gttgtcccaa ttcttgttga
     1981 attagatggt gatgttaacg gccacaagtt ctctgtcagt ggagagggtg aaggtgatgc
     2041 aacatacgga aaacttaccc tgaagttcat ctgcactact ggcaaactgc ctgttccatg
     2101 gccaacacta gtcactactc tgtgctatgg tgttcaatgc ttttcaagat acccggatca
     2161 tatgaaacgg catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaaggac
     2221 catcttcttc aaagatgacg gcaactacaa gacacgtgct gaagtcaagt ttgaaggtga
     2281 tacccttgtt aatagaatcg agttaaaagg tattgacttc aaggaagatg gcaacattct
     2341 gggacacaaa ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca
     2401 aaagaatgga atcaaagtga acttcaagac ccgccacaac attgaagatg gaagcgttca
     2461 actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
     2521 caaccattac ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca
     2581 catggtcctt cttgagtttg taacagctgc tgggattaca catggcagga tgaactgtac
     2641 aactgagaat tctgcagtcg acggtaccgc gggcccggga tccaccgggt acaagtaaag
     2701 cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta
     2761 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     2821 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2881 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2941 taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     3001 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     3061 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     3121 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     3181 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     3241 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     3301 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     3361 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     3421 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3481 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga
     3541 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3601 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3661 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3721 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3781 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     3841 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga
     3901 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3961 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4021 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4081 cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc
     4141 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4201 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     4261 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     4321 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     4381 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     4441 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     4501 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     4561 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4621 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4681 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4741 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4801 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4861 cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg
     4921 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
     4981 cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     5041 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     5101 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     5161 gccatagcct caggttactc atatatactt tagattgatt taaaacttca tttttaattt
     5221 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5281 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5341 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5401 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5461 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     5521 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5581 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5641 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5701 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5761 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5821 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5881 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5941 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6001 gattctgtgg ataaccgtat taccgccatg cat
//
