LOCUS       sequence-10725-e        5727 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 5727)
  AUTHORS   Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, 
            Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar
            EJ
  TITLE     An improved zinc-finger nuclease architecture for highly specific 
            genome editing.
  JOURNAL   Nat Biotechnol. 2007 Jul . 25(7):778-85.
  PUBMED    17603475
REFERENCE   2  (bases 1 to 5727)
  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 
            Biotechnol. 2007 Jul . 25(7):778-85."
COMMENT     Sequence Label: pMLM800
FEATURES             Location/Qualifiers
     source          1..5727
                     /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             991..1575
                     /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="LVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVM
                     EFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRY
                     VKENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVLS
                     VEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             1591..1632
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1642..1659
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1682..1699)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1688..1912
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1958..2386
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2045..2064)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2255..2276
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2395..2415)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2400..2730
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2581..2716
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2643..2662
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2778..2825
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2844..3242
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3400..3521
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3437..3456)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3491..3510
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3570..3586)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3570..3586)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3583..3605)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3594..3610
                     /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(3618..3624,3625..3642,3643..3648))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3663..3684
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3801..3818)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3972..4560)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4052..4071)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4731..5522,5523..5591))
                     /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     5354..5373
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5592..5696)
                     /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 gctgccgctg actacaagga tgacgacgat
      961 aaatctagaa aatatgcggc cgctcgagcc ctagtcaaaa gtgaactgga ggagaagaaa
     1021 tctgaacttc gtcataaatt gaaatatgtg cctcatgaat atattgaatt aattgaaatt
     1081 gccagaaatt ccactcagga tagaattctt gaaatgaagg taatggaatt ttttatgaaa
     1141 gtttatggat atagaggtaa acatttgggt ggatcaagga aaccggacgg agcaatttat
     1201 actgtcggat ctcctattga ttacggtgtg atcgtggata ctaaagctta tagcggaggt
     1261 tataatctgc caattggcca agcagatgaa atgcaacgat atgtcaaaga aaatcaaaca
     1321 cgaaacaaac atatcaaccc taatgaatgg tggaaagtct atccatcttc tgtaacggaa
     1381 tttaagtttt tatttgtgag tggtcacttt aaaggaaact acaaagctca gcttacacga
     1441 ttaaatcata agactaattg taatggagct gttcttagtg tagaagagct tttaattggt
     1501 ggagaaatga ttaaagccgg cacattaacc ttagaggaag tgagacggaa atttaataac
     1561 ggcgagataa acttttaagg gcccttcgaa ggtaagccta tccctaaccc tctcctcggt
     1621 ctcgattcta cgcgtaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca
     1681 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1741 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1801 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     1861 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     1921 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     1981 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2041 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2101 gctctaaatc ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2161 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2221 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2281 acactcaacc ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc
     2341 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     2401 tgtgtcagtt agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc
     2461 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2521 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2581 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2641 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     2701 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     2761 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
     2821 acgacaaggt gaggaactaa accatggcca agcctttgtc tcaagaagaa tccaccctca
     2881 ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca
     2941 gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg
     3001 ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc
     3061 tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt
     3121 gtcgacaggt gcttctcgat ctgcatcctg ggatcaaagc gatagtgaag gacagtgatg
     3181 gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct
     3241 aagcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc
     3301 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3361 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     3421 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3481 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     3541 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3601 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3661 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3721 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3781 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3841 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3901 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3961 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4021 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4081 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4141 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     4201 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4261 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4321 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4381 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     4441 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4501 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4561 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4621 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4681 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4741 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4801 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4861 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4921 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4981 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5041 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5101 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5161 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5221 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5281 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5341 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5401 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5461 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5521 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5581 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5641 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5701 tttccccgaa aagtgccacc tgacgtc
//
