LOCUS       sequence-420377-        5526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of human RPA32 C-terminally fused to EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5526)
  TITLE     Pospiech Lab Plasmids 2023
REFERENCE   2  (bases 1 to 5526)
  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"
COMMENT     Sequence Label: pRPA32-EGFP
FEATURES             Location/Qualifiers
     source          1..5526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        60..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"
     regulatory      1465..1474
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1471..1473,1474..1476,1477..2190)
                     /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             join(1471..1473,1474..1476,1477..2187)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1516..1537)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1777..1796)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2124..2145
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2313..2434
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2350..2369)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2404..2423
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2441..2896)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2578..2599)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2790..2809
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2923..3027
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3029..3386
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3029..3220
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3052..3072)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3237..3372
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3421..4215
                     /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             3421..4215
                     /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(3475..3494)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4085..4104
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4403..4422)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4447..4494
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4823..5411
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5312..5331
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
       61 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      121 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      181 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      241 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      301 atgaccttat gggactttcc tacttggcag tacatctacg 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 cagagctggt ttagtgaacc gtcagatccg ctagcgctac
      601 cggactcaga tctcgagctc aagcttcgaa ttcgccacca tgtggaacag tggattcgaa
      661 agctatggca gctcctcata cgggggagcc ggcggctaca cgcagtcccc ggggggcttt
      721 ggatcgcccg caccttctca agccgaaaag aaatcaagag cccgagccca gcacattgtg
      781 ccctgtacta tatctcagct gctttctgcc actttggttg atgaagtgtt cagaattggg
      841 aatgttgaga tttcacaggt cactattgtg gggatcatca gacatgcaga gaaggctcca
      901 accaacattg tttacaaaat agatgacatg acagctgcac ccatggacgt tcgccagtgg
      961 gttgacacag atgacaccag cagtgaaaac actgtggttc ctccagaaac atatgtgaaa
     1021 gtggcaggcc acctgagatc ttttcagaac aaaaagagcc tggtagcctt taagatcatg
     1081 cccctggagg atatgaatga gttcaccaca catattctgg aagtgatcaa tgcacacatg
     1141 gtactaagca aagccaacag ccagccctca gcagggagag cacctatcag caatccagga
     1201 atgagtgaag cagggaactt tggtgggaat agcttcatgc cagcaaatgg cctcactgtg
     1261 gcccaaaacc aggtgttgaa tttgattaag gcttgtccaa gacctgaagg gttgaacttt
     1321 caggatctca agaaccagct gaaacacatg tctgtatcct caatcaagca agctgtggat
     1381 tttctgagca atgaggggca catctattct actgtggatg atgaccattt taaatccaca
     1441 gatgcagaag cggatccacc ggtcgccacc atggtgagca agggcgagga gctgttcacc
     1501 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1561 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1621 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1681 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1741 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1801 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1861 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1921 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1981 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2041 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2101 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2161 actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc ataatcagcc
     2221 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     2281 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2341 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2401 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     2461 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2521 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2581 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2641 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2701 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2761 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2821 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2881 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2941 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3001 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     3061 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3121 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3181 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3241 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3301 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3361 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     3421 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3481 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3541 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3601 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3661 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3721 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3781 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3841 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3901 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     3961 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4021 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4081 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4141 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4201 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4261 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4321 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4381 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     4441 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4501 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     4561 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     4621 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     4681 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4741 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4801 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4861 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4921 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4981 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5041 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5101 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5161 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5221 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5281 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5341 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5401 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5461 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     5521 tgcatt
//
