LOCUS       sequence-7549-ex        5466 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 5466)
  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 5466)
  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: pc3XB-ZF64
FEATURES             Location/Qualifiers
     source          1..5466
                     /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"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1056..1073)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1062..1286
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1332..1760
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1419..1438)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1629..1650
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1769..1789)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1774..2103
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1954..2089
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2016..2035
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2174..2968
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2228..2247)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2838..2857
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3142..3263
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3179..3198)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3233..3252
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3312..3328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3312..3328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3325..3347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3336..3352
                     /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(3360..3366,3367..3384,3385..3390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3405..3426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3543..3560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3714..4299)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3794..3813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4470..5261,5262..5330))
                     /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     5093..5112
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5331..5435)
                     /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 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga cccggggaga agccttacaa atgcccagaa tgtggaaaga
      961 gttttagcac ttctggaaat ctcgtgagac accagagaac acataccggt ggatcctctg
     1021 cacagtagta agcttaagtt taaaccgctg atcagcctcg actgtgcctt ctagttgcca
     1081 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1141 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     1201 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     1261 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     1321 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1381 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1441 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1501 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1561 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1621 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1681 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1741 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     1801 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     1861 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     1921 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     1981 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     2041 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     2101 aaaaagctcc cggccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     2161 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     2221 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     2281 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     2341 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     2401 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     2461 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     2521 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     2581 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     2641 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     2701 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     2761 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     2821 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     2881 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     2941 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     3001 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     3061 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     3121 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     3181 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3241 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     3301 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3361 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     3421 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3481 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3541 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3601 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3661 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3721 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3781 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3841 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3901 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3961 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4021 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4081 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4141 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4201 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tttttttgtt
     4261 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4321 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4381 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4441 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4501 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4561 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4621 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4681 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4741 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4801 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4861 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4921 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4981 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5041 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5101 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5161 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5221 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5281 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5341 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5401 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5461 gacgtc
//
