LOCUS       sequence-42847-e        6267 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 6267)
  AUTHORS   Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH
  TITLE     Real-time observation of transcription initiation and elongation on 
            an endogenous yeast gene.
  JOURNAL   Science. 2011 Apr 22;332(6028):475-8.
  PUBMED    21512033
REFERENCE   2  (bases 1 to 6267)
  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: "Science."; 
            date: "2011-04-22"; volume: "332(6028)"; pages: "475-8"
COMMENT     Sequence Label: pDZ264 (pKAN tdtomato cyc loxP KAN loxP)
FEATURES             Location/Qualifiers
     source          1..6267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..36)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(21..37)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      39..286
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(55..73)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    337..370
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            425..1781
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        425..768
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             769..1578
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(836..855)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1584..1781
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1844..1877
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1928..1948)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1928..1946)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1967..1983)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1967..1983)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1980..2002)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1991..2007
                     /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(2015..2021,2022..2039,2040..2045))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2060..2081
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2198..2215)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2369..2957)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2449..2468)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3128..3919,3920..3988))
                     /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     3751..3770
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3989..4093)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4120..4575
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4207..4226)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4417..4438
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4701..4723
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4715..4732
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4716..4732
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4742..4761
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4742..4760
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4793..4809
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4793..4809
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      4815..4824
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4821..6248
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
ORIGIN
        1 aattcgatat caagcttatc gataccgtcg acctcgagtc atgtaattag ttatgtcacg
       61 cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc
      121 tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat
      181 atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa
      241 aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccagc
      301 tgaagcttcg tacgctgcag gtcgacaacc cttaatataa cttcgtataa tgtatgctat
      361 acgaagttat taggtctaga gatctgttta gcttgcctcg tccccgccgg gtcacccggc
      421 cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc
      481 atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc
      541 catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg
      601 cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta
      661 gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa
      721 aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaggaa
      781 aagactcacg tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
      841 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
      901 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
      961 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     1021 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca
     1081 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     1141 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     1201 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     1261 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg
     1321 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     1381 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     1441 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     1501 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     1561 ctcgatgagt ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt
     1621 catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt
     1681 atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa
     1741 tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac
     1801 gccgccatcc agtgtcgaaa acgagctctc gagaaccctt aatataactt cgtataatgt
     1861 atgctatacg aagttattag gtgatatcag atccactagt ggcctatgcg gtacccagct
     1921 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     1981 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     2041 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2101 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2161 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2221 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2281 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2341 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2401 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2461 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2521 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2581 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2641 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2701 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2761 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     2821 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2881 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2941 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3001 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3061 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3121 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3181 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3241 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3301 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3361 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3421 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3481 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3541 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3601 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3661 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3721 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3781 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3841 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3901 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3961 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4021 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4081 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt agcggcgcat
     4141 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4201 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4261 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4321 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4381 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4441 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     4501 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4561 taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     4621 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
     4681 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc
     4741 gtaatacgac tcactatagg gcgaattgga gctccaccgc ggtggcggcc gctctagaac
     4801 tagtggatcc ggtcgccacc atggtgagca agggcgagga ggtcatcaaa gagttcatgc
     4861 gcttcaaggt gcgcatggag ggctccatga acggccacga gttcgagatc gagggcgagg
     4921 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc
     4981 ccctgccctt cgcctgggac atcctgtccc cccagttcat gtacggctcc aaggcgtacg
     5041 tgaagcaccc cgccgacatc cccgattaca agaagctgtc cttccccgag ggcttcaagt
     5101 gggagcgcgt gatgaacttc gaggacggcg gtctggtgac cgtgacccag gactcctccc
     5161 tgcaggacgg cacgctgatc tacaaggtga agatgcgcgg caccaacttc ccccccgacg
     5221 gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc
     5281 gcgacggcgt gctgaagggc gagatccacc aggccctgaa gctgaaggac ggcggccact
     5341 acctggtgga gttcaagacc atctacatgg ccaagaagcc cgtgcaactg cccggctact
     5401 actacgtgga caccaagctg gacatcacct cccacaacga ggactacacc atcgtggaac
     5461 agtacgagcg ctccgagggc cgccaccacc tgttcctggg gcatggcacc ggcagcaccg
     5521 gcagcggcag ttccggcacc gcttcttccg aggacaacaa catggccgtc atcaaagagt
     5581 tcatgcgctt caaggtgcgc atggagggct ccatgaacgg ccacgagttc gagatcgagg
     5641 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     5701 gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttcatgtac ggctccaagg
     5761 cgtacgtgaa gcaccccgcc gacatccccg attacaagaa gctgtccttc cccgagggct
     5821 tcaagtggga gcgcgtgatg aacttcgagg acggcggtct ggtgaccgtg acccaggact
     5881 cctccctgca ggacggcacg ctgatctaca aggtgaagat gcgcggcacc aacttccccc
     5941 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctccacc gagcgcctgt
     6001 acccccgcga cggcgtgctg aagggcgaga tccaccaggc cctgaagctg aaggacggcg
     6061 gccgctacct ggtggagttc aagaccatct acatggccaa gaagcccgtg caactgcccg
     6121 gctactacta cgtggacacc aagctggaca tcacctccca caacgaggac tacaccatcg
     6181 tggaacagta cgagcgctcc gagggccgcc accacctgtt cctgtacggc atggacgagc
     6241 tgtacaagtc cggaatatat aagggcg
//
