LOCUS       sequence-106030-        5725 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 5725)
  AUTHORS   Foley JE, Maeder ML, Pearlberg J, Joung JK, Peterson RT, Yeh JR
  TITLE     Targeted mutagenesis in zebrafish using customized zinc-finger 
            nucleases.
  JOURNAL   Nat Protoc. 2009. 4(12):1855-67.
  PUBMED    20010934
REFERENCE   2  (bases 1 to 5725)
  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.
            2009. 4(12):1855-67."
COMMENT     Sequence Label: pMLM292
FEATURES             Location/Qualifiers
     source          1..5725
                     /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"
     CDS             910..930
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             940..963
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             986..1573
                     /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
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMER
                     YVEENQTRNKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             1589..1630
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1640..1657
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1680..1697)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1686..1910
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1956..2384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2043..2062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2253..2274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2393..2413)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2398..2728
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2579..2714
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2641..2660
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2776..2823
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2842..3240
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3398..3519
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3435..3454)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3489..3508
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3568..3584)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3568..3584)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3581..3603)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3592..3608
                     /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(3616..3622,3623..3640,3641..3646))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3661..3682
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3799..3816)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3970..4558)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4050..4069)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4729..5520,5521..5589))
                     /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     5352..5371
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5590..5694)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 accatgggac ctaagaaaaa gaggaaggtg gcggccgctg actacaagga tgacgacgat
      961 aaatctagat agtaagcttg gatcccaact agtcaaaagt gaactggagg agaagaaatc
     1021 tgaacttcgt cataaattga aatatgtgcc tcatgaatat attgaattaa ttgaaattgc
     1081 cagaaattcc actcaggata gaattcttga aatgaaggta atggaatttt ttatgaaagt
     1141 ttatggatat agaggtaaac atttgggtgg atcaaggaaa ccggacggag caatttatac
     1201 tgtcggatct cctattgatt acggtgtgat cgtggatact aaagcttata gcggaggtta
     1261 taatctgcca attggccaag cagatgaaat ggagcgatat gtcgaagaaa atcaaacacg
     1321 aaacaaacat ctcaacccta atgaatggtg gaaagtctat ccatcttctg taacggaatt
     1381 taagttttta tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt
     1441 aaatcatatc actaattgta atggagctgt tcttagtgta gaagagcttt taattggtgg
     1501 agaaatgatt aaagccggca cattaacctt agaggaagtg agacggaaat ttaataacgg
     1561 cgagataaac ttttaagggc ccttcgaagg taagcctatc cctaaccctc tcctcggtct
     1621 cgattctacg cgtaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc
     1681 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     1741 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     1801 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     1861 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     1921 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     1981 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2041 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2101 tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2161 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2221 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2281 actcaaccct atctcggtct attcttttga tttataaggg attttgggga tttcggccta
     2341 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2401 tgtcagttag ggtgtggaaa gtccccaggc tccccaggca ggcagaagta tgcaaagcat
     2461 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     2521 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     2581 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2641 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     2701 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     2761 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     2821 gacaaggtga ggaactaaac catggccaag cctttgtctc aagaagaatc caccctcatt
     2881 gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc
     2941 gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg
     3001 ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg
     3061 acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgt
     3121 cgacaggtgc ttctcgatct gcatcctggg atcaaagcga tagtgaagga cagtgatgga
     3181 cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa
     3241 gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt ccaccgccgc
     3301 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3361 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     3421 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3481 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     3541 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3601 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3661 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3721 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3781 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3841 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3901 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3961 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4021 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4081 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4141 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
     4201 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4261 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4321 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4381 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     4441 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4501 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4561 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4621 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4681 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4741 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4801 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4861 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4921 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4981 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5041 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5101 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5161 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5221 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5281 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5341 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5401 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5461 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5521 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5581 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5641 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5701 tccccgaaaa gtgccacctg acgtc
//
