LOCUS       sequence-259364-        8714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8714)
  AUTHORS   Unsal-Kacmaz K, Mullen TE, Kaufmann WK, Sancar A
  TITLE     Coupling of human circadian and cell cycles by the timeless protein.
  JOURNAL   Mol Cell Biol. 2005 Apr . 25(8):3109-16.
  PUBMED    15798197
REFERENCE   2  (bases 1 to 8714)
  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: "Mol Cell 
            Biol. 2005 Apr . 25(8):3109-16."
COMMENT     Sequence Label: pcDNA4-Flag-Timeless
FEATURES             Location/Qualifiers
     source          1..8714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             731..754
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1751..1762)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4317..4328
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             4403..4432
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4448..4465
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4488..4505)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4494..4718
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4764..5192
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4851..4870)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5061..5082
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5201..5221)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5206..5535
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5386..5521
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5448..5467
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5583..5630
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5649..6023
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    6153..6274
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6190..6209)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6244..6263
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6323..6339)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6323..6339)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6336..6358)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6347..6363
                     /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(6371..6377,6378..6395,6396..6401))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6416..6437
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6554..6571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6725..7313)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6805..6824)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7484..8275,8276..8344))
                     /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     8107..8126
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8345..8449)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8524..8543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga attctgcaga
      721 tatcaggatg gactacaagg acgacgatga caaggacttg cacatgatga actgtgaact
      781 tctagccaca tgtagtgccc ttgggtactt ggagggagac acttaccata aggaaccaga
      841 ttgcttagag agcgtgaagg atctgatccg ctatttgagg catgaggatg agacacgaga
      901 tgtgcggcag cagctggggg cagcccagat cctacagagc gaccttctgc ccatcctcac
      961 ccagcaccac caggacaagc ctctctttga tgctgttatc agactgatgg tgaacttgac
     1021 acaaccagcc ttgctctgtt ttggcaatct gcctaaggag cccagctttc ggcaccattt
     1081 tttgcaggtg ctaacttatt tgcaggccta caaagaggcc tttgccagtg agaaggcttt
     1141 tggagtcctc agtgaaacct tgtatgagct gctgcagctg ggctgggagg aacggcagga
     1201 ggaagacaac ttgctgattg aacggatcct actgctggtc agaaatattc tccatgtccc
     1261 agctgacctt gatcaggaga agaagattga tgatgacgcc agtgcccatg accagctcct
     1321 ctgggcgatt cacctcagcg gcctggatga cctgctcctc tttctggcca gctcgtctgc
     1381 tgaggagcaa tggagcctgc atgtgctaga gattgtctcc cttatgtttc gtgaccagaa
     1441 ccccgagcag ctggcgggag tagggcaggg acgcttagct caggagcgga gtgcagattt
     1501 tgcagaactg gaggtattgc gccagcgaga gatggcagaa aagaagactc gagccctcca
     1561 gcgaggcaac aggcattctc gatttggggg ctcctatatt gtccaggggt tgaaatccat
     1621 tggggagagg gacctcatct ttcacaaagg ccttcacaac ctacgaaact acagttcaga
     1681 tttgggaaag cagccgaaaa aggtgcctaa acgtcgccag gccgcccgag agctgtccat
     1741 tcagcgccgt tctgccctca atgtgaggct cttcctcaga gacttctgct ctgagttcct
     1801 ggagaactgt tacaaccggc tcatgggatc agtaaaggat cacctgcttc gggagaaagc
     1861 tcagcagcat gatgagacct attatatgtg ggccttggct ttcttcatgg ccttcaaccg
     1921 agctgcctcc ttccggccag gcctggtttc tgagaccctc agtgtccgta ccttccactt
     1981 cattgagcag aacctcacca actactatga gatgatgctg actgaccgca aggaagctgc
     2041 ctcctgggca cgccggatgc acttggctct gaaggcctat caggagctgc tggcaacagt
     2101 gaatgagatg gacttatctc cagatgaggc tgtgagggag agcagccgca tcatcaagaa
     2161 caatattttc tatgtgatgg agtaccgaga actattcctg gcactttttc gaaagtttga
     2221 tgagagatgc cagccccgct ctttccttcg tgacctggtg gagaccaccc acctcttcct
     2281 caaaatgttg gagcgattct gtcggagccg tgggaacctg gtggtgcaga acaaacaaaa
     2341 gaagagaagg aagaagaaga agaaggtcct agaccaggcc attgtttctg gtaatgtccc
     2401 atctagccca gaagaagtgg aggctgtgtg gccagccctg gctgagcagc tacagtgctg
     2461 tgcccagaat tctgagctca gcatggactc cgtggttccc tttgatgcgg cctcagaggt
     2521 gccagtggag gagcagcggg cagaagctat ggtacggatc caagactgtc tcctggctgg
     2581 ccaggcccca caggccctga ctctcctgag gtctgctcgg gaggtgtggc ctgaaggaga
     2641 tgtgtttggc tctcaagaca tttctccaga ggaagagatc cagttgctga aacaaatcct
     2701 ctctgctcca cttccccggc agcagggccc agaggaacgt ggggcagagg aagaagaaga
     2761 ggaggaggag gaggaagagg aggagttgca agtggtccag gtgtcggaga aagaatttaa
     2821 ttttctggac tacctgaaac gctttgcatg ttcaactgtc gttcgagcct atgtgctgct
     2881 actaaggagc taccagcaga atagtgccca cactaaccat tgcattgtga agatgctgca
     2941 ccggctggcc catgacctca aaatggaagc cctacttttt cagctgtcag tcttctgcct
     3001 cttcaatcgt ctgcttagtg accctgctgc tggagcctac aaagagctag tgacttttgc
     3061 caaatacatc ctgggcaaat tttttgcact ggctgcagtc aaccaaaaag cctttgtgga
     3121 gctgttgttc tggaagaaca cagctgtggt tcgagagatg actgagggct atggctccct
     3181 ggatgacagg tcttccagtc gcagagcacc tacatggagc cccgaagaag aggctcatct
     3241 tcaggagctg tacctcgcca ataaggacgt ggaagggcag gatgtggtgg aagccatctt
     3301 ggcccacctg aatactgttc ctcgaacacg caagcagatc atccaccatc tggtacagat
     3361 gggactggct gacagtgtca aggacttcca aaggaaagga acccatattg tactgtggac
     3421 gggggatcag gagttggagc tgcagcggct ttttgaggaa ttccgggact cagatgatgt
     3481 cctgggtcat atcatgaaga atatcacagc caaacgctca cgggcccgaa tagtggataa
     3541 actcttggct ctggggctgg tggctgagcg gcgggagctg tacaagaaac ggcagaaaaa
     3601 gttggcatcc tccatcttgc caaatggagc ggagtccctg aaagattttt gccaggaaga
     3661 tctggaagaa gaggaaaacc tgcctgagga agacagcgaa gaggaagaag aagggggctc
     3721 agaagcagaa caagtccagg gtagcttagt cctttcaaat gaaaaccttg gtcaaagcct
     3781 gcatcaggaa ggcttttcta tcccgctcct atggctccag aactgcctga tccgagcagc
     3841 tgatgatcgg gaagaggatg gctgctccca ggccgttcca ttggtgccac tcacagagga
     3901 aaatgaggaa gccatggaaa acgaacagtt tcagcagctg ttgcgcaagc taggggttcg
     3961 gccccctgcc tctgggcagg aaaccttctg gcgaattcca gccaagctga gtcctaccca
     4021 gctccggagg gcagcagctt ctttgagtca accagaggag gaacagaagc tgcagccaga
     4081 gctgcagcct aaagtccctg gagagcaagg ctctgatgag gagcactgta aagagcaccg
     4141 agcacaagcc ctgagggccc tcttgctagc ccacaagaag aaagcgggcc tggcatcccc
     4201 agaggaggaa gacgctgttg gtaaagagcc gctgaaggca gcacccaaga aacgacaatt
     4261 gctggacagc gacgaggaac aggaagaaga tgagggcagg aacagagcac cagagttggg
     4321 agctccagga atccaaaaga agaaacgata ccagattgag gatgatgagg atgagcggcc
     4381 gctcgagtct agagggccct tcgaacaaaa actcatctca gaagaggatc tgaatatgca
     4441 taccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
     4501 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4561 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4621 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4681 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     4741 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4801 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4861 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4921 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4981 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5041 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5101 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5161 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     5221 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     5281 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     5341 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     5401 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     5461 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     5521 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac
     5581 gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga
     5641 actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg
     5701 gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact
     5761 tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg
     5821 tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt
     5881 ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg
     5941 gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact
     6001 tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc gccgccttct
     6061 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     6121 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     6181 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     6241 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     6301 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     6361 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     6421 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6481 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6541 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6601 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6661 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6721 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6781 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6841 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6901 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6961 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     7021 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     7081 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     7141 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     7201 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     7261 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     7321 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     7381 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     7441 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7501 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7561 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7621 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7681 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7741 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7801 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7861 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7921 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7981 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     8041 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     8101 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     8161 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     8221 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     8281 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     8341 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     8401 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8461 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     8521 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     8581 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     8641 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     8701 cagatatacg cgtt
//
