LOCUS       sequence-14312-e        6536 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 6536)
  AUTHORS   Soellu C, Pars K, Cornu TI, Thibodeau-Beganny S, Maeder ML, Joung 
            JK, Heilbronn R, Cathomen T
  TITLE     Autonomous zinc-finger nuclease pairs for targeted chromosomal 
            deletion.
  JOURNAL   Nucleic Acids Res. 2010 Dec 1. 38(22):8269-76.
  PUBMED    20716517
REFERENCE   2  (bases 1 to 6536)
  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: "Nucleic 
            Acids Res. 2010 Dec 1. 38(22):8269-76."
COMMENT     Sequence Label: pMG10
FEATURES             Location/Qualifiers
     source          1..6536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        133..336
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     287..307
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      376..385
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             385..450
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             457..477
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1118..1171
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1178..1243
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1250..1270
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1311..1898
                     /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             1914..1955
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1965..1982
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2005..2022)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2011..2235
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2281..2709
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2368..2387)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2578..2599
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2718..2738)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2723..3053
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2904..3039
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2966..2985
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3101..3148
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3167..3565
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3723..3844
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3760..3779)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3814..3833
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3893..3909)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3893..3909)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3906..3928)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3917..3933
                     /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(3941..3947,3948..3965,3966..3971))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3986..4007
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4124..4141)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4295..4883)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4375..4394)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5054..5845,5846..5914))
                     /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     5677..5696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5915..6019)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6098..6117)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6289..6536,1..132)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
       61 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      121 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      181 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      241 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      301 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc
      361 tagagatccg cggccgccac catggattat aaggatcacg atggcgacta caaggaccac
      421 gatattgact acaaagacga tgacgataaa atggctccta agaaaaagcg caaagtcggt
      481 atccatggcg ttcccgctag ctaacgcaag atctcagctg gtgaagtccg agctcgaaga
      541 aaaaaagagc gagctgcgcc acaagctcaa gtacgtgccc cacgagtaca tcgagctgat
      601 cgagatcgcc cgcaacagca cccaagaccg catcctggag atgaaagtga tggagttctt
      661 catgaaggtg tacggctacc gcggcaagca cctgggcggc tcccgcaagc ccgatggcgc
      721 catctacacc gtgggctccc ccatcgacta tggcgtcatt gtcgacacca aggcctactc
      781 cggcggctac aacttaccca tcggtcaggc cgacgagatg caacgctacg tgaaggagaa
      841 ccagacccgc aataagcaca ttaatcccaa cgagtggtgg aaggtgtacc cctcctccgt
      901 gaccgagttc aaattcctgt tcgtgtccgg ccacttcaag ggcaattata aggcccaact
      961 gacccgcctg aaccacaaga ccaactgcaa cggcgccgtg ctgtccgtgg aggaactgct
     1021 gatcggcggc gagatgatca aggctggtac cctgaccctg gaagaggtgc gccgcaagtt
     1081 caacaatggt gaaatcaatt tcaggtccgg cggcggagag ggcagaggaa gtcttctaac
     1141 atgcggtgac gtggaggaga atcccggccc taggatggac tacaaagacc atgacggtga
     1201 ttataaagat catgacatcg attacaagga tgacgatgac aagatggccc ccaagaagaa
     1261 gaggaaggtg ggcattcacg gggtgccgtc tagatagtaa gcttggatcc caactagtca
     1321 aaagtgaact ggaggagaag aaatctgaac ttcgtcataa attgaaatat gtgcctcatg
     1381 aatatattga attaattgaa attgccagaa attccactca ggatagaatt cttgaaatga
     1441 aggtaatgga attttttatg aaagtttatg gatatagagg taaacatttg ggtggatcaa
     1501 ggaaaccgga cggagcaatt tatactgtcg gatctcctat tgattacggt gtgatcgtgg
     1561 atactaaagc ttatagcgga ggttataatc tgccaattgg ccaagcagat gaaatggagc
     1621 gatatgtcga agaaaatcaa acacgaaaca aacatctcaa ccctaatgaa tggtggaaag
     1681 tctatccatc ttctgtaacg gaatttaagt ttttatttgt gagtggtcac tttaaaggaa
     1741 actacaaagc tcagcttaca cgattaaatc atatcactaa ttgtaatgga gctgttctta
     1801 gtgtagaaga gcttttaatt ggtggagaaa tgattaaagc cggcacatta accttagagg
     1861 aagtgagacg gaaatttaat aacggcgaga taaactttta agggcccttc gaaggtaagc
     1921 ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat caccatcacc
     1981 attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2041 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2101 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2161 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2221 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     2281 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2341 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2401 cgttcgccgg ctttccccgt caagctctaa atcggggcat ccctttaggg ttccgattta
     2461 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2521 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2581 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2641 aagggatttt ggggatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2701 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2761 aggcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2821 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2881 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2941 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3001 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3061 cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc
     3121 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagccttt
     3181 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat
     3241 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg
     3301 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc
     3361 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg
     3421 catcttgagc ccctgcggac ggtgtcgaca ggtgcttctc gatctgcatc ctgggatcaa
     3481 agcgatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc
     3541 ctctggttat gtgtgggagg gctaagcact tcgtggccga ggagcaggac tgacacgtgc
     3601 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3661 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     3721 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3781 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3841 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     3901 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3961 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4021 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4081 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4141 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4201 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4261 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4321 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4381 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4441 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct
     4501 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4561 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4621 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4681 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4741 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4801 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4861 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4921 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4981 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5041 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5101 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5161 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5221 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5281 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5341 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5401 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5461 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5521 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5581 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5641 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5701 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5761 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5821 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5881 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5941 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6001 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     6061 gagatcgatc tcccgatccc ctatggtcga ctctcagtac aatctgctct gatgccgcat
     6121 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     6181 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     6241 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     6301 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     6361 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     6421 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     6481 gtcaatgggt ggactattta cggtaaactg cccacttggc agtacatcaa gtgtat
//
