LOCUS       sequence-7639-ex        5528 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 5528)
  AUTHORS   Wright DA, Thibodeau-Beganny S, Sander JD, Winfrey RJ, Hirsh AS, 
            Eichtinger M, Fu F, Porteus MH, Dobbs D, Voytas DF, Joung JK
  TITLE     Standardized reagents and protocols for engineering zinc finger 
            nucleases by modular assembly.
  JOURNAL   Nat Protoc. 2006;1(3):1637-52. doi: 10.1038/nprot.2006.259
  PUBMED    17406455
REFERENCE   2  (bases 1 to 5528)
  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: "Nat 
            Protoc."; date: "2006"; volume: "1(3):1637-52. doi"; pages: " 
            10.1038/nprot.2006.25"
COMMENT     Sequence Label: pDW1775
FEATURES             Location/Qualifiers
     source          1..5528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      36..130
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     137..156
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        137..155
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      214..300
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        390..569
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             656..676
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             677..703
                     /codon_start=1
                     /product="epitope tag from the baculovirus Autographa 
                     californica GP64 envelope fusion protein"
                     /label=AcV5 tag
                     /translation="SWKDASGWS"
     CDS             726..1313
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     terminator      1336..1588
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        complement(1630..1648)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      complement(1650..1689)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(1989..2648)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(2649..2836)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     misc_feature    2987..3490
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        3532..3636
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4704..5292
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5193..5212
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5446..5463
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtcaacgaca tcacctggtt gcgagccttc tgaaggactc ctgttgatag atccagtaat
       61 gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat
      121 tggtgagaat caggcgtaat acgactcact atagggcgaa tggaactgga cgctcgcctg
      181 tggtcgcagg gtggctcgta cgaccgcagg catcaaataa aacgaaaggc tcagtcgaaa
      241 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
      301 ccgccagcgg atgagcggag aattaaggga gtcacgttat gacccccgcc gatgacgcgg
      361 gacaagccgt tttacgtttg gaactgacag aaccgcaacg ttgaaggagc cactcagccg
      421 ccggtttctg gagtttaatg agctaagcac atacgtcaga aaccattatt gcgcgttcaa
      481 aagtcgccta aggtcactat caggtagcaa atatttcttg tcaaaaatgc tccactgacg
      541 ttccataaat tcccctcggt atccaattag aaaggtggga ctcacgaaga tctaactcga
      601 gaaagatatt gtatatatcg taacaatagg aggttcaaca atggcttcct cccctccaaa
      661 gaaaaagaga aaggttagtt ggaaggacgc aagtggttgg tctagacccg ggaaccggtg
      721 gatcccagct agtgaaatct gaattggaag agaagaaatc tgaacttaga cataaattga
      781 aatatgtgcc acatgaatat attgaattga ttgaaatcgc aagaaattca actcaggata
      841 gaatccttga aatgaaggtg atggagttct ttatgaaggt ttatggttat cgtggtaaac
      901 atttgggtgg atcaaggaaa ccagacggag caatttatac tgtcggatct cctattgatt
      961 acggtgtgat cgttgatact aaggcatatt caggaggtta taatcttcca attggtcaag
     1021 cagatgaaat gcaaagatat gtcgaagaga atcaaacaag aaacaagcat atcaacccta
     1081 atgaatggtg gaaagtctat ccatcttcag taacagaatt taagttcttg tttgtgagtg
     1141 gtcatttcaa aggaaactac aaagctcagc ttacaagatt gaatcatatc actaattgta
     1201 atggagctgt tcttagtgta gaagagcttt tgattggtgg agaaatgatt aaagctggta
     1261 cattgacact tgaggaagtg agaaggaaat ttaataacgg tgagataaac ttttaatagg
     1321 agctcgaatt tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct
     1381 gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata
     1441 attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa
     1501 ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg
     1561 cgcgcggtgt catctatgtt actagatcgt gggcccacat tcctgtagaa ctcggtgaga
     1621 cttttgttcc ctttagtgag ggttaattgt ccaaaaaaaa aggctccaaa aggagccttt
     1681 aattgtttcg gtttatgctt aggacaccat tgattaacgc ttacaatttc ctgatgcggt
     1741 attttctcct tacgcatctg tgcggtattt cacaccgcat agatccgtcg agttcaagag
     1801 aaaaaaaaag aaaaagcaaa aagaaaaaag gaaagcgcgc ctcgttcaga atgacacgta
     1861 tagaatgatg cattaccttg tcatcttcag tatcatactg ttcgtataaa tacttactga
     1921 cattcatagc tatacatata tacacatgta tatatatcgt atgctggagc tttaaataat
     1981 cggtgtcact acataagaac acctttggtg gagggaacat cgttggttcc attgggcgag
     2041 gtggcttctc ttatggcaac cgcaagagcc ttgaacgcac tctcactacg gtgatggtca
     2101 ttcttgcctc gcagacaatc aacgtggagg gtaattctgc ttgcctctgc aaaactttca
     2161 agaaaatgcg ggatcatctc gcaagagagg tctcctactt tctccctttg caaaccaagt
     2221 tcgacaactg cgtatggcct gttagaaagg tctaccaccg ctctggaaag tgcctcatcc
     2281 aaaggcgcaa atcctgatcc aaaccttttt actccacgcg ccagtagagc ctctttaaaa
     2341 gcctgaccga gagcaatccc gcagtcttca gtggtgtgat ggtcgtctat gtgtaagtca
     2401 ccaatgcact caacgattag cgaccagccg gaatgcttgg ccagagcatg tatcatgtgg
     2461 tccagaaacc ctatacctgt gtggacgtta atcacttgcg attgtgtggc ctgttctgct
     2521 actgcttctg cctctttttc tgggaagatc gagtgctcta tcgctagggg accacccttt
     2581 aaagagatag caatctgaat cttggtttca tttgtaatac gctttactag ggctttctgc
     2641 tctgtcatct ttgccttcgt ttatcttgcc tgctcatttt ttagtatatt cttcgaagaa
     2701 atcacattac tttatataat gtataattca ttatgtgata atgccaatcg ctaagaaaaa
     2761 aaaagagtca tccgctaggt ggaaaaaaaa aaatgaaaat cattaccgag gcataaaaaa
     2821 atatagagtg tactagagga ggccaagagt aatagaaaaa gaaaattgcg ggaaaggact
     2881 gtgttatgac ttccctgact aatgccgtgt tcaaacgata cctggcagtg actcctagcg
     2941 ctcaccaagc tcttaaaacg ggaatttatg gatcttaatt aagacggatc gcttgcctgt
     3001 aacttacacg cgcctcgtat cttttaatga tggaataatt tgggaattta ctctgtgttt
     3061 atttattttt atgttttgta tttggatttt agaaagtaaa taaagaaggt agaagagtta
     3121 cggaatgaag aaaaaaaaat aaacaaaggt ttaaaaaatt tcaacaaaaa gcgtacttta
     3181 catatatatt tattagacaa gaaaagcaga ttaaatagat atacattcga ttaacgataa
     3241 gtaaaatgta aaatcacagg attttcgtgt gtggtcttct acacagacaa gatgaaacaa
     3301 ttcggcatta atacctgaga gcaggaagag caagataaaa ggtagtattt gttggcgatc
     3361 cccctagagt cttttacatc ttcggaaaac aaaaactatt ttttctttaa tttctttttt
     3421 tactttctat ttttaattta tatatttata ttaaaaaatt taaattataa ttatttttat
     3481 agcacgtgat gaaaagcctt agggtcaggt ggcacttttc ggggaaatgt gcgatgaacc
     3541 cctatttgtt tatttttcta aatacattca aatatgtatc cgctaatgag acaataaccc
     3601 tgataaatgc ttcaattata ttgaaaaagg aagagtatga gtatccaaca tttccgtgtc
     3661 gcccttattc ccttttttgc ggccttctgc cttcctgttt ttgctcaccc agaaacgctg
     3721 gtgaaagtaa aagatgctga agatcagttg ggtgcccgag tgggttacat tgaactggat
     3781 ctcaacagcg gcaaaatctt agagagtttt cgccccgaag aacgcttccc aatgatgagc
     3841 actttcaaag ttctgctatg tggcgcggtg ttatcccgta ttgatgctgg gcaagagcaa
     3901 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtattcacc agtcacagaa
     3961 aagcatctta cggatggcat gacagtaaga gaattatgta gtgctgccat aaccatgagt
     4021 gataacactg cggccaactt acttctgaca acgattggag gaccgaagga gctaaccgct
     4081 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4141 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaactctc
     4201 cgcaaactat taactggcga actacttact cttgcttcac gccagcaact cattgactgg
     4261 atggaggcgg acaaagttgc aggaccactt ctgcgctcgg cacttccggc tggctggttt
     4321 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4381 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4441 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4501 tcagacgaag ggaaataaat agtagcccgc ctgatgtgcg ggcttttttt tggtggtggt
     4561 taccagtgtc gcttagctac cgtaggtcct cttaggcggt ccgattagcg acatagtcat
     4621 caatgccttg acggcaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4681 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4741 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctactaact
     4801 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4861 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4921 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4981 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgctca
     5041 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5101 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5161 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5221 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5281 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5341 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5401 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     5461 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5521 tgatgcag
//
