LOCUS       sequence-7530-ex        5469 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 5469)
  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 5469)
  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-ZF131
FEATURES             Location/Qualifiers
     source          1..5469
                     /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(1059..1076)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1065..1289
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1335..1763
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1422..1441)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1632..1653
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1772..1792)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1777..2106
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1957..2092
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2019..2038
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2177..2971
                     /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(2231..2250)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2841..2860
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3145..3266
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3182..3201)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3236..3255
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3315..3331)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3315..3331)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3328..3350)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3339..3355
                     /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(3363..3369,3370..3387,3388..3393))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3408..3429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3546..3563)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3717..4302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3797..3816)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4473..5264,5265..5333))
                     /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     5096..5115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5334..5438)
                     /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 agcctttcct gtgccagtat tgcgcccagc
      961 gtttcggtcg caaggaccac ttaacgcgcc acatgaagaa gagccatacc ggtggatcct
     1021 ctgcacagta gtaagcttaa gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg
     1081 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     1141 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     1201 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     1261 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     1321 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1381 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1441 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1501 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1561 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1621 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1681 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1741 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     1801 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     1861 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     1921 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     1981 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2041 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     2101 tgcaaaaagc tcccggccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     2161 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2221 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     2281 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     2341 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     2401 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     2461 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     2521 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     2581 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     2641 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     2701 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     2761 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     2821 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     2881 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     2941 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     3001 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3061 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3121 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3181 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3241 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3301 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3361 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3421 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3481 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3541 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3601 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3661 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3721 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3781 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3841 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3901 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3961 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4021 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4081 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4141 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4201 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggttttttt
     4261 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4321 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4381 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4441 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4501 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4561 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4621 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4681 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4741 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4801 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4861 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4921 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4981 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5041 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5101 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5161 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5221 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5281 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     5341 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     5401 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     5461 cctgacgtc
//
