LOCUS       sequence-202458-        5068 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 5068)
  AUTHORS   Scot Wolfe
  TITLE     Inducible Cas9-DNA-binding-domain fusions for precise genome editing
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5068)
  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            "Unpublished"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..5068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             193..471
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             877..942
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             976..1002
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             1048..1068
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1113..1247
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1137..1156)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1191..1210
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1364..1384)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1364..1382)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1403..1419)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1403..1419)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1416..1438)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1427..1443
                     /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(1451..1457,1458..1475,1476..1481))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1496..1517
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1634..1651)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1805..2393)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1885..1904)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2564..3355,3356..3424))
                     /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     3187..3206
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3425..3529)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3555..4010)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3692..3713)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3904..3923
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4084..5068
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atctgccacc atgggtctcg atttagcttc attaattcta
      121 ggtcctggct cacttgacct cgctagtctt atcctcggag gcggtggcag cggcggaggt
      181 ggctcacgta cgatcctctg gcatgagatg tggcatgaag gcctggaaga ggcatctcgt
      241 ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg tgctggagcc cttgcatgct
      301 atgatggaac ggggccccca gactctgaag gaaacatcct ttaatcaggc ctatggtcga
      361 gatttaatgg aggcccaaga gtggtgcagg aagtacatga aatcagggaa tgtcaaggac
      421 ctcacccaag cctgggacct ctattatcat gtgttccgac gaatctcaaa gggcgccggc
      481 ggaagcgggg taccttccgg aaagccctat aaatgtcctg aatgtggcaa gtccttctcg
      541 cggagcgacg acctgacacg gcaccaacgt acgcacactg gtgagaagcc atacgcgtgt
      601 cctgtcgagt cctgtgaccg ccgcttcagt cagaagggac acctgacacg gcacatccgc
      661 attcacacag ggcaaaaacc gtttcaatgc cgcatctgca tgaggaactt cagcatccgt
      721 agcagcctga cacggcacat ccgcacccac acaggagaaa agcccttcgc ctgtgacatc
      781 tgcggcagga agttcgcgct gagccaccac ctgacacggc acaccaagat ccacctccgt
      841 cagaaagacc ccgggggatc cgggtcgctc gagggagact acaaagacca tgacggtgat
      901 tataaagatc atgacatcga ttacaaggat gacgatgaca agcaggggtc cgagctcgga
      961 tcgggtagag ccgcgccggc agctaagaaa aagaaactgg atcaattggg cgctagcggt
     1021 agttctggag gctcaagtgg cgccgctcca aagaagaagc gtaaggtatc tgaattctga
     1081 taatctagaa ctatagtgag tcgtattacg tagatccaga catgataaga tacattgatg
     1141 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     1201 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     1261 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcgcggccg
     1321 cggcgccaat gcattgggcc cggtacgtac ccagcttttg ttccctttag tgagggttaa
     1381 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     1441 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     1501 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     1561 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     1621 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     1681 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1741 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1801 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1861 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1921 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1981 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2041 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2101 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2161 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2221 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     2281 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2341 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2401 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     2461 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2521 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2581 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     2641 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     2701 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     2761 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     2821 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     2881 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     2941 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3001 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3061 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3121 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3181 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3241 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3301 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3361 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3421 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3481 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     3541 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     3601 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     3661 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     3721 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     3781 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     3841 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3901 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3961 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     4021 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     4081 gtcgaccata gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat
     4141 ctggacctgt gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat
     4201 ctggacccca gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac
     4261 ttggcactgt gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt
     4321 cttggccctg tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata
     4381 ttggctatat gccaggatca atatataggc aatatccaat atggccctat gccaatatgg
     4441 ctattggcca ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg
     4501 ttttcctatt gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc
     4561 ttcctaatac cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt
     4621 tcctattgac gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc
     4681 aattacggta aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca
     4741 ttgacgtcaa tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg
     4801 tcaatgacgg taaatggccc acttggcagt acatcaatat ctattaatag taacttggca
     4861 agtacattac tattggaagg acgccagggt acattggcag tactcccatt gacgtcaatg
     4921 gcggtaaatg gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc
     4981 aatgacgcaa atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt
     5041 ctatataagc aatgctcgtt tagggaac
//
