LOCUS       sequence-204643-        5065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ZFP-VEGFA-TS3 fused to FRB in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5065)
  AUTHORS   Scot Wolfe
  TITLE     Inducible Cas9-DNA-binding-domain fusions for precise genome editing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5065)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCSD_2xNES-FRB-ZFPTS3-3xFLAG-2xNLS
FEATURES             Location/Qualifiers
     source          1..5065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        30..48
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     30..47
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      80..89
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             188..466
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             872..937
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             971..997
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             1043..1063
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1108..1242
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1132..1151)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1186..1205
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1359..1379)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1359..1377)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1398..1414)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1398..1414)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1411..1433)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1422..1438
                     /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(1446..1452,1453..1470,1471..1476))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1491..1512
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1629..1646)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1800..2388)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1880..1899)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2559..3350,3351..3419))
                     /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     3182..3201
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3420..3524)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3550..4005)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3687..3708)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3899..3918
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4076..5060
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 ttctgcctgg ggacgtcgga gcaagcttga tttaggtgac actatagaat acaagctact
       61 tgttcttttt gcaggatctg ccaccatggg tctcgattta gcttcattaa ttctaggtcc
      121 tggctcactt gacctcgcta gtcttatcct cggaggcggt ggcagcggcg gaggtggctc
      181 acgtacgatc ctctggcatg agatgtggca tgaaggcctg gaagaggcat ctcgtttgta
      241 ctttggggaa aggaacgtga aaggcatgtt tgaggtgctg gagcccttgc atgctatgat
      301 ggaacggggc ccccagactc tgaaggaaac atcctttaat caggcctatg gtcgagattt
      361 aatggaggcc caagagtggt gcaggaagta catgaaatca gggaatgtca aggacctcac
      421 ccaagcctgg gacctctatt atcatgtgtt ccgacgaatc tcaaagggcg ccggcggaag
      481 cggggtacct tccggaaagc cctataaatg tcctgaatgt ggcaagtcct tctcgcggag
      541 cgacgacctg acacggcacc aacgtacgca cactggtgag aagccatacg cgtgtcctgt
      601 cgagtcctgt gaccgccgct tcagtcagaa gggacacctg acacggcaca tccgcattca
      661 cacagggcaa aaaccgtttc aatgccgcat ctgcatgagg aacttcagca tccgtagcag
      721 cctgacacgg cacatccgca cccacacagg agaaaagccc ttcgcctgtg acatctgcgg
      781 caggaagttc gcgctgagcc accacctgac acggcacacc aagatccacc tccgtcagaa
      841 agaccccggg ggatccgggt cgctcgaggg agactacaaa gaccatgacg gtgattataa
      901 agatcatgac atcgattaca aggatgacga tgacaagcag gggtccgagc tcggatcggg
      961 tagagccgcg ccggcagcta agaaaaagaa actggatcaa ttgggcgcta gcggtagttc
     1021 tggaggctca agtggcgccg ctccaaagaa gaagcgtaag gtatctgaat tctgataatc
     1081 tagaactata gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt
     1141 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     1201 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     1261 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaattcgc ggccgcggcg
     1321 ccaatgcatt gggcccggta cgtacccagc ttttgttccc tttagtgagg gttaattgcg
     1381 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1441 ccacacaaca tacgagccgg gagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1501 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1561 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1621 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1681 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1741 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1801 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1861 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1921 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1981 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2041 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2101 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2161 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2221 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2281 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2341 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     2401 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2461 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2521 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2581 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2641 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     2701 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     2761 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     2821 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     2881 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     2941 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3001 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3061 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3121 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3181 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3241 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3301 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3361 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3421 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3481 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3541 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     3601 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3661 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3721 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3781 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3841 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3901 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3961 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca
     4021 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagtcga
     4081 ccgccaattc aatatggcgt atatggactc atgccaattc aatatggtgg atctggacct
     4141 gtgccaattc aatatggcgt atatggactc gtgccaattc aatatggtgg atctggaccc
     4201 cagccaattc aatatggcgg acttggcacc atgccaattc aatatggcgg acctggcact
     4261 gtgccaactg gggaggggtc tacttggcac ggtgccaagt ttgaggaggg gtcttggccc
     4321 tgtgccaagt ccgccatatt gaattggcat ggtgccaata atggcggcca tattggctat
     4381 atgccaggat caatatatag gcaatatcca atatggccct atgccaatat ggctattggc
     4441 caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta
     4501 ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat
     4561 accgcccata gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg
     4621 acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg
     4681 taaatggccc gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc
     4741 aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac
     4801 ggtaaatggc ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt
     4861 actattggaa gtacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa
     4921 tggcccgcga tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc
     4981 aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa
     5041 gcaatgctcg tttagggaac cgcca
//
