LOCUS       sequence-419599-        7897 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses shRNA resistant GFP-RepoMan T394A Mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7897)
  AUTHORS   Pfisterer M, Robert R, Saul VV, Pritz A, Seibert M, Feederle R, 
            Schmitz ML
  TITLE     The Aurora B-controlled PP1/RepoMan complex determines the spatial 
            and temporal distribution of mitotic H2B S6 phosphorylation.
  JOURNAL   Open Biol. 2024 May;14(5):230460. doi: 10.1098/rsob.230460. Epub 
            2024 May 29.
  PUBMED    38806145
REFERENCE   2  (bases 1 to 7897)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Open 
            Biol."; date: "2024-05"; volume: "14(5):230460. doi"; pages: " 
            10.1098/rsob.230460. Epub 2024 May 29"
COMMENT     Sequence Label: pEGFP-RepoMan-T394A
FEATURES             Location/Qualifiers
     source          1..7897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4459..4548
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     polyA_signal    4685..4806
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4722..4741)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4776..4795
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4813..5268)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4950..4971)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5162..5181
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5295..5399
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5401..5758
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5401..5592
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5424..5444)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5609..5744
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5793..6587
                     /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             5793..6587
                     /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(5847..5866)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6457..6476
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6775..6794)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6819..6866
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7195..7783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7684..7703
                     /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 gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc
     1381 cgccggcgaa tggatgccaa ttcaaaagac aagccccctg aaaccaagga gtctgcaatg
     1441 aataatgctg gaaatgcctc tttcattttg ggaactggga agattgtgac tcctcagaag
     1501 catgccgaat tacctcctaa tccttgcaca ccagatactt ttaaatcacc tttgaacttt
     1561 tccacagtaa ccgtagagca attgggaatt acacctgaaa gctttgttag gaactctgca
     1621 ggaaagtcat catcctacct taaaaaatgt agacgacgtt ctgcagtcgg tgctcggggc
     1681 tctcctgaaa caaaccatct gattcgtttc attgctcggc agcaaaatat aaagaatgct
     1741 aggaaatctc ctttggcaca agattctcct tcccagggca gccctgcact gtatcgaaat
     1801 gttaacactt taagagaacg aatatcagcc ttccagtcag cttttcactc cataaaggaa
     1861 aacgagaaaa tgaccggctg tctggaattc tcagaggcag gaaaagagtc cgagatgacc
     1921 gatctgacaa ggaaggaagg tctcagcgct tgccagcagt ctgggttccc tgcagtgttg
     1981 tcctccaaac gtcggagaat atcctatcag agagactctg atgaaaatct gacggatgct
     2041 gaaggaaaag taattggtct ccagatattc aatattgata cagacagagc atgtgcagtt
     2101 gaaacttctg tcgaccttag cgaaatttca agcaagcttg gttcaacaca gtctggattt
     2161 ttagttgaag agtctcttcc cctttcagag ctcacagaga cttcaaatgc actaaaggtt
     2221 gctgactgtg tagtgggcaa aggatcaagt gatgccgttt cgcctgacac gttcacagca
     2281 gaagtgagct cagacgcagt ccctgatgtc aggtcaccag ctactccagc ctgcaggagg
     2341 gaccttccca cccccaagac ctttgtactt cgttctgtac tgaagaaacc ctctgttaag
     2401 atgtgtctag agagcttaca ggaacactgt aacaacctct atgatgatga tgggactcat
     2461 ccgagcttaa tctcaaatct cccaaactgt tgcaaagaga aagaagcaga agatgaagaa
     2521 aattttgaag cacctgcctt tctaaatatg aggaagagga agagagttgc ttttggagag
     2581 gacttaagcc cggaagtgtt tgatgaatct ttgccagcaa atactccatt gcgtaaagga
     2641 ggaacacctg tttgtaaaaa agacttcagt ggtctcagtt ccctgctgct tgagcagtca
     2701 cctgttcctg agccattacc tcaaccagat tttgatgaca agggggagaa tcttgaaaac
     2761 atagaaccac ttcaagtatc atttgccgtt ctcagttctc ctaataaatc atcaatctct
     2821 gagacccttt caggcactga tacctttagt tcttcaaata accatgagaa aatatcctca
     2881 ccaaaggtgg gccgtatcac aaggacttct aaccggagga accagctggt tagcgtggta
     2941 gaagaaagcg tgtgtaatct cctgaacaca gaagttcagc cttgtaaaga aaagaaaatt
     3001 aataggagga agtctcaaga aacaaagtgt acaaagagag cacttcctaa gaagagtcag
     3061 gttttaaaaa gttgcagaaa gaagaaagga aagggaaaga aaagtgttca gaaatcttta
     3121 tatggggaaa gagacattgc ttctaagaag cccctcctca gtcctattcc cgagctgcct
     3181 gaagtccctg agatgacacc ttccattccg agcatccgaa gactgggttc aggttatttc
     3241 agttcaaatg gcaaactgga agaagtgaag actcctaaaa atccagtgaa aagaaaggat
     3301 cttttgcgtc atgacccaga tttgcatatg catcaaggct atgataaata tgatgtctct
     3361 gaattctgct cttatataaa aagttcctca tcgcttggca atgctacttc tgatgaggac
     3421 ccgaacacta acattatgaa tataaacgag aacaaaaata ttccaaaagc aaaaaataag
     3481 tcagaaagtg aaaatgaacc aaaagctgga actgacagtc ctgtttcttg tgcttctgta
     3541 actgaagaac gtgtggcatc agatagtccc aaacctgctc tgaccctgca gcagggtcaa
     3601 gaattttctg ctggtggtca aaatgcagaa aacctttgtc agttctttaa aatttcacca
     3661 gatttaaaca taaagtgtga aagaaaggat gacttcttag gagctgcaga aggaaaactg
     3721 caatgcaatc gtttaatgcc taattcacaa aaagactgtc attgtttagg agatgtctta
     3781 attgaaaata cgaaagaatc taaaagccag agtgaggatt tgggaagaaa acccatggaa
     3841 agtagcagtg ttgtgagttg cagagacagg aaagatagaa gacgttccat gtgttattct
     3901 gatggtcgaa gtttacattt ggaaaaaaat ggaaatcaca caccatcctc cagtgtgggc
     3961 agctctgtag aaattagttt agaaaattct gaactgttta aagatttgtc tgatgccatt
     4021 gagcaaacct ttcagaggag aaatagtgaa accaaagtgc gacgtagcac gaggctacag
     4081 aaggatttag aaaacgaagg tcttgtatgg atttcacttc cacttccttc cacttcccaa
     4141 aaagccaaaa gaagaacaat atgtacattt gacagcagtg gatttgaaag tatgtctccc
     4201 ataaaagaaa ctgtgtcctc cagacaaaaa ccgcagatgg cacctcccgt ctcagatcca
     4261 gaaaacagcc agggccctgc tgctggttct tccgatgaac ctggtaagag gaggaagagc
     4321 ttttgtatat ctacacttgc aaatactaaa gccacttccc agttcaaagg ctaccggaga
     4381 agatcctctc ttaatgggaa gggagagagc tctctgactg ccttggaaag gattgaacat
     4441 aatggagaaa gaaagcagta cccatacgat gttcctgact atgcgggcta tccgtatgac
     4501 gtcccggact atgcaggatc ctatccatat gacgttccag attacgctta gcccgggatc
     4561 caccggatct agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc
     4621 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     4681 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4741 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4801 atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     4861 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     4921 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     4981 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     5041 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     5101 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     5161 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     5221 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     5281 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5341 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     5401 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     5461 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     5521 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     5581 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     5641 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     5701 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     5761 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     5821 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     5881 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     5941 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     6001 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     6061 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     6121 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     6181 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     6241 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     6301 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     6361 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     6421 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     6481 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     6541 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     6601 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     6661 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     6721 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     6781 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     6841 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     6901 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     6961 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     7021 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     7081 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     7141 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7201 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7261 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7321 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7381 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7441 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7501 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7561 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7621 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7681 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7741 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7801 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7861 ccctgattct gtggataacc gtattaccgc catgcat
//
