LOCUS       sequence-460685-        6748 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Human RPA34 tagged to GFP under a EF1alpha promotor for generating 
            stable HeLa Kyoto GFP-RPA34 using the Flp-In recombination system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6748)
  AUTHORS   Pabba MK, Ritter C, Chagin VO, Meyer J, Celikay K, Stear JH, Loerke 
            D, Kolobynina K, Prorok P, Schmid AK, Leonhardt H, Rohr K, Cardoso 
            MC
  TITLE     Replisome loading reduces chromatin motion independent of DNA 
            synthesis.
  JOURNAL   Elife. 2023 Oct 31;12:RP87572. doi: 10.7554/eLife.87572.
  PUBMED    37906089
REFERENCE   2  (bases 1 to 6748)
  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: "Elife."; 
            date: "2023-10-31"; volume: "12:RP87572. doi"; pages: " 
            10.7554/eLife.87572"
COMMENT     Sequence Label: pFRT-B-GRPA
FEATURES             Location/Qualifiers
     source          1..6748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        351..1529
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          582..1520
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1477..1497
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1546..1565
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1546..1564
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1639..1659
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1672..1681
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1678..1680,1681..1683,1684..2394)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1678..1680,1681..1683,1684..2394)
                     /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(1723..1744)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1984..2003)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2331..2352
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3323..3364
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3402..3419)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3408..3632
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3765..3784)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3916..3963
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3977..4372
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4414..4535
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4451..4470)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4505..4524
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4591..4607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4591..4607)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4604..4626)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4615..4631
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4639..4645,4646..4663,4664..4669))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4684..4705
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4822..4839)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4993..5581)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5073..5092)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5752..6543,6544..6612))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6375..6394
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6613..6717)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac taggcttttg caaaaagctt tgcaaagatg gataaagttt taaacagaga
      301 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      361 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      421 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      481 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      541 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      601 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      661 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      721 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      781 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      841 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      901 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      961 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
     1021 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
     1081 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1141 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1201 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1261 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1321 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1381 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1441 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1501 gtttttttct tccatttcag gtgtcgtgag gaattagctt ggtactaata cgactcacta
     1561 tagggagacc caagctggct aggtaagctt ggtaccgagc tcggatcatc ccttcaccat
     1621 atggcttcgt ggggatcccc gaagaagaag cgcaaagtac tggtaccggt cgccaccatg
     1681 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1741 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1801 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1861 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1921 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1981 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2041 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2101 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2161 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2221 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2281 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2341 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga
     2401 ctcagatctc gagctcaagc ttcgaattct atgtggaaca gtggattcga aagctatggc
     2461 agctcctcat acgggggagc cggcggctac acgcagtccc cggggggctt tggatcgccc
     2521 gcaccttctc aagccgaaaa gaaatcaaga gcccgagccc agcacattgt gccctgtact
     2581 atatctcagc tgctttctgc cactttggtt gatgaagtgt tcagaattgg gaatgttgag
     2641 atttcacagg tcactattgt ggggatcatc agacatgcag agaaggctcc aaccaacatt
     2701 gtttacaaaa tagatgacat gacagctgca cccatggacg ttcgccagtg ggttgacaca
     2761 gatgacacca gcagtgaaaa cactgtggtt cctccagaaa catatgtgaa agtggcaggc
     2821 cacctgagat cttttcagaa caaaaagagc ctggtagcct ttaagatcat gcccctggag
     2881 gatatgaatg agttcaccac acatattctg gaagtgatca atgcacacat ggtactaagc
     2941 aaagccaaca gccagccctc agcagggaga gcacctatca gcaatccagg aatgagtgaa
     3001 gcagggaact ttggtgggag tagcttcatg ccagcaaatg gcctcactgt ggcccaaaac
     3061 caggtgttga atttgattaa ggcttgtcca agacctgaag ggttgaactt tcaggatctc
     3121 aagaaccagc tgaaacacat gtctgtatcc tcaatcaagc aagctgtgga ttttctgagc
     3181 aatgaggggc acatctattc tactgtggat gatgaccatt ttaaatccac agatgcagaa
     3241 taattctagt ccagtgtggt ggaattctgc agatatccag cacagtggcg gccgctcgag
     3301 tctagagggc ccgcggttcg aaggtaagcc tatccctaac cctctcctcg gtctcgattc
     3361 tacgcgtacc ggttagtaat gagtttaaac ccgctgatca gcctcgactg tgccttctag
     3421 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3481 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3541 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3601 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     3661 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3721 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3781 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3841 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3901 tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac
     3961 ttccttggaa ttcaaggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
     4021 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
     4081 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
     4141 tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
     4201 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
     4261 gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     4321 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct aagcacttcg
     4381 tggccgagga gcaggactga cactcgacct cgaaacttgt ttattgcagc ttataatggt
     4441 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4501 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgaattcc gtataccgtc
     4561 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4621 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4681 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4741 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4801 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4861 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4921 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4981 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5041 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5101 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5161 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5221 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5281 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5341 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5401 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5461 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5521 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5581 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5641 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5701 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5761 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5821 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5881 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5941 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6001 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6061 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6121 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6181 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6241 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6301 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6361 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6421 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6481 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6541 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6601 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6661 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6721 atttccccga aaagtgccac ctgacgtc
//
