LOCUS       sequence-7475-ex        5472 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 5472)
  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 5472)
  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-ZF27
FEATURES             Location/Qualifiers
     source          1..5472
                     /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(1062..1079)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1068..1292
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1338..1766
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1425..1444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1635..1656
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1775..1795)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1780..2109
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1960..2095
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2022..2041
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2180..2974
                     /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(2234..2253)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2844..2863
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3148..3269
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3185..3204)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3239..3258
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3318..3334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3318..3334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3331..3353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3342..3358
                     /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(3366..3372,3373..3390,3391..3396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3411..3432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3549..3566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3720..4305)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3800..3819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4476..5267,5268..5336))
                     /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     5099..5118
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5337..5441)
                     /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 cccggggagc ggccatttat gtgtacctgg tcatactgtg
      961 ggaaacgctt cacacgtagc gatcacctga cacgtcacaa acgtacacac accggtggat
     1021 cctctgcaca gtagtaagct taagtttaaa ccgctgatca gcctcgactg tgccttctag
     1081 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     1141 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     1201 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     1261 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     1321 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1381 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1441 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1501 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1561 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1621 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1681 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1741 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     1801 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     1861 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     1921 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     1981 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2041 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2101 ttttgcaaaa agctcccggc cgggagcttg tatatccatt ttcggatctg atcaagagac
     2161 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     2221 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     2281 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     2341 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     2401 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     2461 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     2521 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     2581 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     2641 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     2701 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     2761 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     2821 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     2881 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     2941 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     3001 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     3061 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     3121 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     3181 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3241 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     3301 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3361 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3421 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3481 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3541 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3601 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3661 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3721 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3781 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3841 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3901 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3961 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4021 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4081 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4141 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4201 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt
     4261 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4321 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4381 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4441 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4501 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4561 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4621 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4681 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4741 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4801 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4861 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4921 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4981 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5041 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5101 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5161 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5221 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5281 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5341 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5401 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5461 ccacctgacg tc
//
