LOCUS       sequence-106028-        5724 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 5724)
  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 5724)
  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: pMLM290
FEATURES             Location/Qualifiers
     source          1..5724
                     /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
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVKENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVL
                     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..2727
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2578..2713
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2640..2659
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2775..2822
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2841..3239
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3397..3518
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3434..3453)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3488..3507
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3567..3583)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3567..3583)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3580..3602)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3591..3607
                     /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(3615..3621,3622..3639,3640..3645))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3660..3681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3798..3815)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3969..4557)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4049..4068)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4728..5519,5520..5588))
                     /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     5351..5370
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5589..5693)
                     /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 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 gcaacgatat gtcaaagaaa atcaaacacg
     1321 aaacaaacat atcaacccta atgaatggtg gaaagtctat ccatcttctg taacggaatt
     1381 taagttttta tttgtgagtg gtcactttaa aggaaactac aaagctcagc ttacacgatt
     1441 aaatcataag 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 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2161 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2221 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2281 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2341 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2401 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2461 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2521 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     2581 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     2641 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     2701 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     2761 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     2821 acaaggtgag gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg
     2881 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg
     2941 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg
     3001 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga
     3061 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc
     3121 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac
     3181 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag
     3241 cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc
     3301 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     3361 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     3421 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3481 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     3541 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3601 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3661 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3721 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3781 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3841 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3901 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3961 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4021 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4081 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4141 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4201 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4261 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4321 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4381 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4441 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4501 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4561 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4621 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4681 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4741 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4801 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4861 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4921 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4981 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5041 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5101 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5161 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5221 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5281 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5341 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5401 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5461 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5521 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5581 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5641 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5701 ccccgaaaag tgccacctga cgtc
//
