LOCUS       sequence-12767-e        5763 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 5763)
  AUTHORS   Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, 
            Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, 
            Unger-Wallace E, Sander JD, Muller-Lerch F, Fu F, Pearlberg J, 
            Goebel C, Dassie JP, Pruett-Miller SM, Porteus MH, Sgroi DC, Iafrate
            AJ, Dobbs D, McCray PB, Cathomen T, Voytas DF, Joung JK
  TITLE     Rapid ""open-source"" engineering of customized zinc-finger 
            nucleases for highly efficient gene modification.
  JOURNAL   Mol Cell. 2008 Jul 25. 31(2):294-301.
  PUBMED    18657511
REFERENCE   2  (bases 1 to 5763)
  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: "Mol Cell. 
            2008 Jul 25. 31(2):294-301."
COMMENT     Sequence Label: CACNA1F_L (OZ507)
FEATURES             Location/Qualifiers
     source          1..5763
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(12..17,18..35,36..42)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    50..66
                     /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)."
     CDS             368..391
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     gap             446..457
                     /estimated_length=12
     gap             530..541
                     /estimated_length=12
     gap             614..625
                     /estimated_length=12
     rep_origin      793..1221
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(880..899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1090..1111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1555..1574)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1566..2381
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1566..2381
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1639..1658)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(4010..4026)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4034..4050
                     /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(4058..4064,4065..4082,4083..4088))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(4332..4350)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4441..5029
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4930..4949
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5183..5200
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5215..5355
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5281..5300)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5441..5463
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5457..5648)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gaattccggg ctttacactt tatgcttccg gctcgtataa tgtgtcgact tgtgagcgga
       61 taacaatttc acacaggaaa cagctatgcc tcaacagcag caaatgcaac ctcccaattc
      121 aagtgcggac aacaaccctt tgcaacagca atcatcacaa aataccgtac caaacgtcct
      181 caaccaaatt aaccaaatct tttctccaga ggagcaacgc agcttattac aagaagccat
      241 cgaaacctgc aagaattttg aaaaaacaca attgggtagt acgatgacgg aacctgtcaa
      301 gcaaagtttt attaggaaat acattgtgca aaaggccctg agaaaaatcc aagctttggc
      361 ggccgctgac tacaaagacg atgatgacaa atctagaccc ggggagcgcc ccttccagtg
      421 tcgcatttgc atgcggaact tttcgnnnnn nnnnnnnctt nnnnnncata cccgtactca
      481 taccggtgaa aaaccgtttc agtgtcggat ctgtatgcga aatttctccn nnnnnnnnnn
      541 nttgnnnnnn catctacgta cgcacaccgg cgagaagcca ttccaatgcc gaatatgcat
      601 gcgcaacttc agtnnnnnnn nnnnnctgnn nnnncaccta aaaacccacc tgaggggatc
      661 ctctgcacag tagtaactag agactagaaa aaggccgaca agtcccgctc cgctgaagat
      721 cctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      781 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      841 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      901 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      961 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1021 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1081 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1141 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1201 aacaaaaatt taacgcgaat tttaacaaaa tctagcgctg aggtctgcct cgtgaagaag
     1261 gtgttgctga ctcataccag gcctgaatcg ccccatcatc cagccagaaa gtgagggagc
     1321 cacggttgat gagagctttg ttgtaggtgg accagttggt gattttgaac ttttgctttg
     1381 ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg atccttcaac tcagcaaaag
     1441 ttcgatttat tcaacaaagc cacgttgtgt ctcaaaatct ctgatgttac attgcacaag
     1501 ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     1561 gtgttatgag ccatattcaa cgggaaacgt cttgctcgag gccgcgatta aattccaaca
     1621 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     1681 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     1741 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     1801 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     1861 ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     1921 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     1981 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     2041 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     2101 ggaaagaaat gcataagctt ttgccattct caccggattc agtcgtcact catggtgatt
     2161 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     2221 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     2281 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     2341 ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt
     2401 tgtaacactg gcagagcatt acgctgactt gacgggacgg cggctttgtt gaataaatcg
     2461 aacttttgct gagttgaagg atcagatcac gcatcttccc gacaacgcag accgttccgt
     2521 ggcaaagcaa aagttcaaaa tcaccaactg gtccacctac aacaaagctc tcatcaaccg
     2581 tggctccctc actttctggc tggatgatgg ggcgattcag gcctggtatg agtcagcaac
     2641 accttcttca cgaggcagac ctcagcgctc aaagatgcag gggtaaaagc taaccgcatc
     2701 tttaccgaca aggcatccgg cagttcaaca gatcgggaag ggctggattt gctgaggatg
     2761 aaggtggagg aaggtgatgt cattctggtg aagaagctcg accgtcttgg ccgcgacacc
     2821 gccgacatga tccaactgat aaaagagttt gatgctcagg gtgtagcggt tcggtttatt
     2881 gacgacggga tcagtaccga cggtgatatg gggcaaatgg tggtcaccat cctgtcggct
     2941 gtggcacagg ctgaacgccg gaggatcagc catggatcat ttattctgcc tcccagagcc
     3001 tgataaaaac ggttagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     3061 gcatcctgcg atgcagatcc taggtcaaaa taatcctgtt aacaatgctt ttatatcctg
     3121 taaagaatcc attttcaaaa tcatgtcaag gtcctcccga ggaaaaatca gtagaaatag
     3181 ctgttccagt ctttctagcc ttgattctgc ggccgcctct ggtttctctt ctttcacttc
     3241 aggctgacgt acatcacgta agtcaacgaa agcttccagt tgttcagcca gaatggttgc
     3301 cgcacgacga atcgcctctt caggatcgat tgtgccgttg gtttccattt cgatgaccag
     3361 cttgtccagg tcggtacgct gttctacacg cgctgcttca acattgtagg caatacgctc
     3421 cacagggctg tagcatgcgt cgaccagcag acggccgatt gggcgctcat cttcttccga
     3481 atgaattcgg gtagaagccg gcacataacc acgaccgcgc tgaactttga tacgcatgct
     3541 aatagacgcg ttctcatcgg tcaggtggca gatcacgtgc tgcggcttga cgatttcgac
     3601 atcaccgtcg tgggtgatat cggctgcagt cacagggcca atgccagatt tattcaaggt
     3661 aagaataact tcatctttgc cctgaactct caccgccagc cctttcaggt tgagcaggat
     3721 ttccaggata tcttcctgaa cgccttcttt ggtgctgtac tcatgtagta caccatcaat
     3781 ctcaacctcg gtcaccgcgc aacccggcat cgatgagagc agaatacggc gcagtgcgtt
     3841 acccagagta tggccaaagc cacgctctaa aggctcaagg gtcaccttgg cgtgcgtcga
     3901 actcacttgc tcgatatcaa ccaggcgcgg ttttagaaac tctgtcacag aaccctgcat
     3961 tgtgtcctct ctttggtcta gctagagttc cagtgaatcc gtaatcatgg tcatagctgt
     4021 ttcctgtgtg aaattgttat ccgctcacaa ttccacacat tatacgagcc ggaagcataa
     4081 agtgtaaagc ctctctagct agattgagtt aatctccatg tagcgttaca agatctgaag
     4141 gggttcgaat tctatttttt tgatgggaag gcacttattt ttgatcgttc gctcaaagaa
     4201 gcatcgaacg ctgggtttta gataaaaaaa ttgagagaaa acaggatgct tccatcggat
     4261 tcatcttaga taaagtgaga ttatatagtg aaatccgatt tgatttttta ttgcttctgg
     4321 ttatccaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga ccaaaatccc
     4381 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4441 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4501 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4561 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4621 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4681 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4741 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4801 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4861 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4921 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4981 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5041 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5101 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     5161 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     5221 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     5281 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5341 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5401 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5461 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5521 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5581 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5641 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5701 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5761 cgg
//
