LOCUS       sequence-204639-        5065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses  ZFP-VEGFA-TS2 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  JOURNAL   Unpublished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            "Unpublished"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-ZFPTS2-3xFLAG-2xNLS
FEATURES             Location/Qualifiers
     source          1..5065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..54
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     36..53
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      86..95
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             194..472
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             878..943
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             977..1003
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             1049..1069
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1114..1248
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1138..1157)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1192..1211
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1365..1385)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1365..1383)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1404..1420)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1404..1420)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1417..1439)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1428..1444
                     /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(1452..1458,1459..1476,1477..1482))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1497..1518
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1635..1652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1806..2394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1886..1905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2565..3356,3357..3425))
                     /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     3188..3207
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3426..3530)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3556..4011)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3693..3714)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3905..3924
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(4082..5065,1)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 ccgccattct gcctggggac gtcggagcaa gcttgattta ggtgacacta tagaatacaa
       61 gctacttgtt ctttttgcag gatctgccac catgggtctc gatttagctt cattaattct
      121 aggtcctggc tcacttgacc tcgctagtct tatcctcgga ggcggtggca gcggcggagg
      181 tggctcacgt acgatcctct ggcatgagat gtggcatgaa ggcctggaag aggcatctcg
      241 tttgtacttt ggggaaagga acgtgaaagg catgtttgag gtgctggagc ccttgcatgc
      301 tatgatggaa cggggccccc agactctgaa ggaaacatcc tttaatcagg cctatggtcg
      361 agatttaatg gaggcccaag agtggtgcag gaagtacatg aaatcaggga atgtcaagga
      421 cctcacccaa gcctgggacc tctattatca tgtgttccga cgaatctcaa agggcgccgg
      481 cggaagcggg gtaccttccg gaaagcccta taaatgtcct gaatgtggca agtccttctc
      541 ggagaagagc cacctgacac ggcaccaacg cacgcacact ggtgagaagc catacgcgtg
      601 tcctgtcgag tcctgtgacc gccgcttcag tcggagcgac cacctgacac agcacatccg
      661 cattcacaca gggcaaaaac cgtttcaatg ccgcatctgc atgaggaact tcagcgacaa
      721 gggccacctg acccggcaca tccgcaccca cacaggagaa aagcccttcg cctgtgacat
      781 ctgcggcagg aagttcgcgc ggagcgacga cctgacacgg cacaccaaga tccacctccg
      841 tcagaaagac cccgggggat ccgggtcgct cgagggagac tacaaagacc atgacggtga
      901 ttataaagat catgacatcg attacaagga tgacgatgac aagcaggggt ccgagctcgg
      961 atcgggtaga gccgcgccgg cagctaagaa aaagaaactg gatcaattgg gcgctagcgg
     1021 tagttctgga ggctcaagtg gcgccgctcc aaagaagaag cgtaaggtat ctgaattctg
     1081 ataatctaga actatagtga gtcgtattac gtagatccag acatgataag atacattgat
     1141 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     1201 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1261 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcgcggcc
     1321 gcggcgccaa tgcattgggc ccggtacgta cccagctttt gttcccttta gtgagggtta
     1381 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     1441 acaattccac acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga
     1501 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1561 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1621 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1681 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1741 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1801 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1861 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1921 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1981 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2041 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2101 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2161 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2221 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2281 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2341 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2401 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2461 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2521 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2581 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2641 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2701 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2761 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2821 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2881 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2941 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3001 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3061 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3121 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3181 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3241 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3301 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3361 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3421 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3481 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3541 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     3601 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     3661 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     3721 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     3781 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     3841 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     3901 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     3961 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     4021 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     4081 agtcgaccgc caattcaata tggcgtatat ggactcatgc caattcaata tggtggatct
     4141 ggacctgtgc caattcaata tggcgtatat ggactcgtgc caattcaata tggtggatct
     4201 ggaccccagc caattcaata tggcggactt ggcaccatgc caattcaata tggcggacct
     4261 ggcactgtgc caactgggga ggggtctact tggcacggtg ccaagtttga ggaggggtct
     4321 tggccctgtg ccaagtccgc catattgaat tggcatggtg ccaataatgg cggccatatt
     4381 ggctatatgc caggatcaat atataggcaa tatccaatat ggccctatgc caatatggct
     4441 attggccagg ttcaatacta tgtattggcc ctatgccata tagtattcca tatatgggtt
     4501 ttcctattga cgtagatagc ccctcccaat gggcggtccc atataccata tatggggctt
     4561 cctaataccg cccatagcca ctcccccatt gacgtcaatg gtctctatat atggtctttc
     4621 ctattgacgt catatgggcg gtcctattga cgtatatggc gcctccccca ttgacgtcaa
     4681 ttacggtaaa tggcccgcct ggctcaatgc ccattgacgt caataggacc acccaccatt
     4741 gacgtcaatg ggatggctca ttgcccattc atatccgttc tcacgccccc tattgacgtc
     4801 aatgacggta aatggcccac ttggcagtac atcaatatct attaatagta acttggcaag
     4861 tacattacta ttggaagtac gccagggtac attggcagta ctcccattga cgtcaatggc
     4921 ggtaaatggc ccgcgatggc tgccaagtac atccccattg acgtcaatgg ggaggggcaa
     4981 tgacgcaaat gggcgttcca ttgacgtaaa tgggcggtag gcgtgcctaa tgggaggtct
     5041 atataagcaa tgctcgttta gggaa
//
