LOCUS       sequence-436283-        8842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding GFP tagged human Timeless.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8842)
  AUTHORS   Prorok P, Wolf E, Cardoso MC
  TITLE     Timeless-Tipin interactions with MCM and RPA mediate DNA replication
            stress response.
  JOURNAL   Front Cell Dev Biol. 2024 Feb 29;12:1346534. doi: 
            10.3389/fcell.2024.1346534. eCollection 2024.
  PUBMED    38487270
REFERENCE   2  (bases 1 to 8842)
  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"; journalName: "Front Cell 
            Dev Biol."; date: "2024-02-29"; volume: "12:1346534. doi"; pages: " 
            10.3389/fcell.2024.1346534. eCollection 2024"
COMMENT     Sequence Label: pEGFP-hTimeless (pc4729)
FEATURES             Location/Qualifiers
     source          1..8842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      866..1001
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1333..1352
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    1351..1406
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1452..1471
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     intron          1603..2175
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     protein_bind    2200..2211
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     CDS             complement(2247..2258)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             4813..4824
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(join(5854..6564,6565..6567,6568..6570))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(5854..6564,6565..6567,6568..6570))
                     /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(5896..5917)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6245..6264
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6504..6525
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      6567..6576
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(6589..6609)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     5'UTR           6645..6684
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     primer_bind     complement(6753..6777)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(6774..6977)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6803..6823)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6978..7281
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(7304..7323)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7305..7323)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7442..7461)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7561..7583
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7621..7639)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7707..7811
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7812..7880,7881..8672)
                     /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     complement(8030..8049)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcaatt agtcagcaac
      841 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
      901 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
      961 tgagctattc cagaagtagt gaagaggctt ttttggaggc ctaggctttt gcaaaaagct
     1021 ccgggatctc cataagagaa gagggacagc tatgactggg agtagtcagg agaggaggaa
     1081 aaatctggct agtaaaacat gtaaggaaaa ttttagggat gttaaagaaa aaaataacac
     1141 aaaacaaaat ataaaaaaaa tctaacctca agtcaaggct tttctatgga ataaggaatg
     1201 gacagcaggg ggctgtttca tatactgatg acctctttat agccaacctt tgttcatggc
     1261 agccagcata tgggcatatg ttgccaaact ctaaaccaaa tactcattct gatgttttaa
     1321 atgatttgcc ctcccatatg tccttccgag tgagagacac aaaaaattcc aacacactat
     1381 tgcaatgaaa ataaatttcc tttattagcc agaagtcaga tgctcaaggg gcttcatgat
     1441 gtccccataa tttttggcag agggaaaaag atctcagtgg tatttgtgag ccagggcatt
     1501 ggccacacca gccaccacct tctgataggc agcctgcacc tgaggagtga attctttgcc
     1561 aaaatgatga gacagcacaa caaccagcac gttgcccagg agctgtagga aagagaagaa
     1621 ggcatgaaca tggttagcag aggggcccgg tttggactca gagtatttta tcctcatctc
     1681 aaacagtgta tatcattgta accataaaga gaaaggcagg atgatgacca gggtgtagtt
     1741 gtttctacca ataagaatat ttccacgcca gccagaattt atatgcagaa atattctacc
     1801 ttatcattta attataacaa ttgttctcta aaactgtgct gaagtacaat ataatatacc
     1861 ctgattgcct tgaaaaaaaa gtgattagag aaagtactta caatctgaca aataaacaaa
     1921 agtgaattta aaaattcgtt acaaatgcaa gctaaagttt aacgaaaaag ttacagaaaa
     1981 tgaaaagaaa ataagaggag acaatggttg tcaacagagt agaaagtgaa agaaacaaaa
     2041 ttatcatgag ggtccatggt gatacaaggg acatcttccc attctaaaca acaccctgaa
     2101 aactttgccc cctccatata acatgaattt tacaatagcg aaaaagaaag aacaatcaag
     2161 ggtccccaaa ctcaccctga agttctcagc tctagatcag tcatcctcat catcctcaat
     2221 ctggtatcgt ttcttctttt ggattcctgg agctcccaac tctggtgctc tgttcctgcc
     2281 ctcatcttct tcctgttcct cgtcgctgtc cagcaattgt cgtttcttgg gtgctgcctt
     2341 cagcggctct ttaccaacag cgtcttcctc ctctggggat gccaggcccg ctttcttctt
     2401 gtgggctagc aagagggccc tcagggcttg tgctcggtgc tctttacagt gctcctcatc
     2461 agagccttgc tctccaggga ctttaggctg cagctctggc tgcagcttct gttcctcctc
     2521 tggttgactc aaagaagctg ctgccctccg gagctgggta ggactcagct tggctggaat
     2581 tcgccagaag gtttcctgcc cagaggcagg gggccgaacc cctagcttgc gcaacagctg
     2641 ctgaaactgt tcgttttcca tggcttcctc attttcctct gtgagtggca ccaatggaac
     2701 ggcctgggag cagccatcct cttcccgatc atcagctgct cggatcaggc agttctggag
     2761 ccataggagc aggatagaaa agccttcctg atgcaggctt tgaccaaggt tttcatttga
     2821 aaggactaag ctaccctgga cttgttctgc ttctgagccc ccttcttctt cctcttcgct
     2881 gtcttcctca ggcaggtttt cctcttcttc cagatcttcc tggcaaaaat ctttcaggga
     2941 ctccgctcca tttggcaaga tggaggatgc caactttttc tgccgtttct tgtacagctc
     3001 ccgccgctca gccaccagcc ccagagccaa gagtttatcc actattcggg cccgtgagcg
     3061 tttggctgtg atattcttca tgatatgacc caggacatca tctgagtccc ggaattcctc
     3121 aaaaagccgc tgcagctcca actcctgatc ccccgtccac agtacaatat gggttccttt
     3181 cctttggaag tccttgacac tgtcagccag tcccatctgt accagatggt ggatgatctg
     3241 cttgcgtgtt cgaggaacag tattcaggtg ggccaagatg gcttccacca catcctgccc
     3301 ttccacgtcc ttattggcga ggtacagctc ccgaagatga gcctcttctt cggggctcca
     3361 tgtaggtgct ctgcgactgg aagacctgtc atccagggag ccatagccct cagtcatctc
     3421 tcgaaccaca gctgtgttct tccagaacaa cagctccaca aaggcttttt ggttgactgc
     3481 agccagtgca aaaaatttgc ccaggatgta tttggcaaaa gtcactagct ctttgtaggc
     3541 tccagcagca gggtcactaa gcagacgatt gaagaggcag aagactgaca gctgaaaaag
     3601 tagggcttcc attttgaggt catgggccag ccggtgcagc atcttcacaa tgcaatggtt
     3661 agtgtgggca ctattctgct ggtagctcct tagtagcagc acataggctc gaacgacagt
     3721 tgaacatgca aagcgtttca ggtagtccag aaaattaaat tctttctccg acacctggac
     3781 cacttgcaac tcctcctctt cctcctcctc ctcctcttct tcttcctctg ccccacgttc
     3841 ctctgggccc tgctgccggg gaagtggagc agagaggatt tgtttcagca actggatctc
     3901 ttcctctgga gaaatgtctt gagagccaaa cacatctcct tcaggccaca cctcccgagc
     3961 agacctcagg agagtcaggg cctgtggggc ctggccagcc aggagacagt cttggatccg
     4021 taccatagct tctgcccgct gctcctccac tggcacctct gaggccgcat caaagggaac
     4081 cacggagtcc atgctgagct cagaattctg ggcacagcac tgtagctgct cagccagggc
     4141 tggccacaca gcctccactt cttctgggct agatgggaca ttaccagaaa caatggcctg
     4201 gtctaggacc ttcttcttct tcttccttct cttcttttgt ttgttctgca ccaccaggtt
     4261 cccacggctc cgacagaatc gctccaacat tttgaggaag aggtgggtgg tctccaccag
     4321 gtcacgaagg aaagagcggg gctggcatct ctcatcaaac tttcgaaaaa gtgccaggaa
     4381 tagttctcgg tactccatca catagaaaat attgttcttg atgatgcggc tgctctccct
     4441 cacagcctca tctggagata tgtccatctc attcactgtt gccagcagct cctgataggc
     4501 cttcagagcc aagtgcatcc ggcgtgccca ggaggcagct tccttgcggt cagtcagcat
     4561 catctcatag tagttggtga ggttctgctc aatgaagtgg aaggtacgga cactgagggt
     4621 ctcagaaacc aggcctggcc ggaaggaggc agctcggttg aaggccatga agaaagccaa
     4681 ggcccacata taataggtct catcatgctg ctgagctttc tcccgaagca ggtgatcctt
     4741 tactgatccc atgagccggt tgtaacagtt ctccaggaac tcagagcaga agtctctgag
     4801 gaagagcctc acattgaggg cagaacggcg ctgaatggac agctctcggg cggcctggcg
     4861 acgtttaggc acctttttcg gctgctttcc caaatctgaa ctgtagtttc gtaggttgtg
     4921 aaggcctttg tgaaagatga ggtccctctc cccaatggat ttcaacccct ggacaatata
     4981 ggagccccca aatcgagaat gcctgttgcc tcgctggagg gctcgagtct tcttttctgc
     5041 catctctcgc tggcgcaaca cctccagttc tgcaaaatct gcactccgct cctgagctaa
     5101 gcgtccctgc cctactcccg ccagctgctc ggggttctgg tcacgaaaca taagggagac
     5161 aatctctagc acatgtaggc tccattgctc ctcagcagac gagctggcca gaaagaggag
     5221 caggtcatcc aggccgctga ggtgaatcgc ccagaggagc tggtcatggg cactggcgtc
     5281 atcatcaatc ttcttctcct gatcaaggtc agctgggaca tggagaatat ttctgaccag
     5341 cagtaggatc cgttcaatca gcaagttgtc ttcctcctgc cgttcctccc agcccagctg
     5401 cagcagctca tacaaggttt cactgaggac tccaaaagcc ttctcactgg caaaggcctc
     5461 tttgtaggcc tgcaaataag ttagcacctg caaaaaatgg tgccgaaagc tgggctcctt
     5521 aggcagattg ccaaaacaga gcaaggctgg ttgtgtcaag ttcaccatca gtctgataac
     5581 agcatcaaag agaggcttgt cctggtggtg ctgggtgagg atgggcagaa ggtcgctctg
     5641 taggatctgg gctgccccca gctgctgccg cacatctcgt gtctcatcct catgcctcaa
     5701 atagcggatg agatccttca cgctctctaa gcaatctggt tccttatggt aagtgtctcc
     5761 ctccaagtac ccaagggcac tacatgtggc tagaagttca cagttcatca tgtgcaagtc
     5821 catcgaagct tgagctcgag atctgagtcc ggacttgtac agctcgtcca tgccgagagt
     5881 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     5941 tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc
     6001 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt
     6061 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
     6121 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
     6181 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
     6241 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
     6301 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca
     6361 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
     6421 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
     6481 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
     6541 ggtgaacagc tcctcgccct tgctcaccat ggtggcgacc ggtaccagta ctttgcgctt
     6601 cttcttcggg gatccccacg aagccatacg cgcttctaca aggcgctggc cgaagaggtg
     6661 cgggagtttc acgccaccaa gatcccccgg aggctggatc ggtcccggtg tcttctatgg
     6721 aggtcaaaac agcgtggatg gcgtctccag gcgatctgac ggttcactaa acgagctctg
     6781 cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg
     6841 ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca
     6901 atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac
     6961 tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta
     7021 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
     7081 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
     7141 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
     7201 acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca
     7261 tttaccgtaa gttatgtaac ggggcgagct cgaattccgg tctccctata gtgagtcgta
     7321 ttaatttcga taagccagac cattccttgc ggcggcggtg ctcaacggcc tcaacctact
     7381 actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgaatgg tgcactctca
     7441 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     7501 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     7561 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     7621 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     7681 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     7741 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     7801 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     7861 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     7921 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     7981 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     8041 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     8101 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     8161 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     8221 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     8281 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     8341 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     8401 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     8461 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     8521 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     8581 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     8641 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     8701 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     8761 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     8821 tagaaaagat caaaggatct tc
//
