LOCUS       pFC544                  4022 bp    DNA     circular SYN 14-AUG-2020
DEFINITION  cHS4-[gRNA1]miniCMV-Gal4-rTa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4022)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2025 from SnapGene 8.1.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    13..18
                     /label=BBb scar
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    complement(25..30)
                     /label=BBb scar
                     /note="color: #a6acb3; direction: LEFT"
     misc_feature    complement(95..955)
                     /label=amp marker
                     /note="Geneious type: marker"
                     /note="color: #a6acb3; direction: LEFT"
     misc_feature    98..955
                     /label=AmpR
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    793..798
                     /label=BBb scar
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    complement(810..815)
                     /label=BBb scar
                     /note="color: #a6acb3; direction: LEFT"
     promoter        complement(997..1025)
                     /label=amp prom
                     /note="color: #ffffff; direction: LEFT"
     misc_feature    1102..1146
                     /label=seq1
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1147..1724
                     /label=pBjh_3260 (concatenated sequence 2)
                     /note="Geneious type: Concatenated sequence"
                     /note="/Source"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1147..1150
                     /label=pL0 I
                     /note="Geneious type: gg_overhang"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1147..1150
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1147..1150
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3"
     misc_feature    1147..1150
                     /label=ligation
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1151..1720
                     /label=2x cHS4 Insulator
                     /note="Geneious type: Insulator"
                     /note="/created_by=User"
                     /note="/modified_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     protein_bind    1229..1233
                     /label=CTCF Bind (Short)
                     /note="/created_by=User"
                     /note="color: #31849b; direction: RIGHT"
     protein_bind    1508..1512
                     /label=CTCF Bind (Short)
                     /note="/created_by=User"
                     /note="color: #31849b; direction: RIGHT"
     misc_feature    1721..1950
                     /label=PCR861 Correct (concatenated sequence 3)
                     /note="Geneious type: Concatenated sequence"
                     /note="/Source"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1721..1724
                     /label=TACT
                     /note="Geneious type: protein_bind"
                     /note="/Transferred_From=pBjh_4574"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/created_by=User"
                     /note="/modified by=User"
                     /note="color: #a6acb3"
     misc_feature    1721..1724
                     /label=pL0 I-P
                     /note="Geneious type: gg_overhang"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1721..1724
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1721..1724
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3"
     misc_feature    1725..1744
                     /label=gRNA BS
                     /note="Geneious type: motif"
                     /note="/created by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1745..1747
                     /label=PAM
                     /note="Geneious type: motif"
                     /note="/created by=User"
                     /note="/modified by=User"
                     /note="color: #a6acb3"
     misc_feature    1766..1785
                     /label=gRNA BS
                     /note="Geneious type: motif"
                     /note="/created by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1786..1788
                     /label=PAM
                     /note="Geneious type: motif"
                     /note="/created by=User"
                     /note="/modified by=User"
                     /note="color: #a6acb3"
     misc_feature    1807..1826
                     /label=gRNA BS
                     /note="Geneious type: motif"
                     /note="/created by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1827..1829
                     /label=PAM
                     /note="Geneious type: motif"
                     /note="/created by=User"
                     /note="/modified by=User"
                     /note="color: #a6acb3"
     misc_feature    1848..1867
                     /label=gRNA BS
                     /note="Geneious type: motif"
                     /note="/created by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1868..1870
                     /label=PAM
                     /note="Geneious type: motif"
                     /note="/created by=User"
                     /note="/modified by=User"
                     /note="color: #a6acb3"
     gene            1871..1946
                     /label=miniCMV
                     /note="/created by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     promoter        1871..1933
                     /label=miniCMV
                     /note="/created by=User"
                     /note="/modified by=User"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    1929
                     /label=Transcription starting point
                     /note="Geneious type: DNA probe bind"
                     /note="/Sequence=A"
                     /note="/created by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1947..1974
                     /label=L0.5.2_Inert (concatenated sequence 4)
                     /note="Geneious type: Concatenated sequence"
                     /note="/Source"
                     /note="color: #a6acb3; direction: RIGHT"
     unsure          1947..1950
                     /label=CAGA
                     /note="/created by=fabio"
                     /note="color: #a6acb3"
     misc_feature    1947..1950
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3"
     misc_feature    1947..1950
                     /label=ligation
                     /note="color: #a6acb3; direction: RIGHT"
     5'UTR           1951..1970
                     /label=Inert
                     /note="color: #a6acb3; direction: RIGHT"
     primer_bind     complement(1958..1980)
                     /label=FCS1
                     /note="color: black; sequence: GGTGGCACCTCTCTTTTTTGTAC"
     misc_feature    1971..3034
                     /label=pBjh_4070 (concatenated sequence 5)
                     /note="Geneious type: Concatenated sequence"
                     /note="/Source"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1971..1974
                     /label=pL0 5-G
                     /note="Geneious type: gg_overhang"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1971..1974
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1971..1974
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3"
     misc_feature    1971..1974
                     /label=ligation
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    1975..1980
                     /label=Kozak
                     /note="Geneious type: motif"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     primer_bind     1979..2002
                     /label=JH1716F
                     /note="color: black; sequence: 
                     ATGGGGTACCCATACGACGTGCCAGATTACGCCATGAAGCTACTGTCCTCTATCG"
     CDS             1981..3030
                     /label=Gal4-NLS-Rta
                     /note="/created_by=User"
                     /note="/modified_by=User"
                     /note="color: #993366; direction: RIGHT"
     misc_feature    1984..2421
                     /label=Gal4 DBD
                     /note="Geneious type: domain"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    2011..2094
                     /label=Zn(2)-C6 fungal-type.{ECO:0000255|PROSITE-ProRu...
                     /note="Geneious type: dna_binding_domain"
                     /note="/Transferred_From=Gal4 DBD (pBjh_4067)"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/created_by=User"
                     /note="/modified_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     primer_bind     2086..2104
                     /label=FCS2
                     /note="color: black; sequence: TGGGAGTGTCGCTACTCTC"
     misc_feature    2422..2448
                     /label=NLS
                     /note="Geneious type: motif"
                     /note="/Transferred_From=Digestion of pRJ_00009 PCR Product
                     - Fragment 2, Digestion of pRJ_00010 PCR Product - Fragment
                     2, Digestion of pRJ_00011 PCR Product - Fragment 2, 
                     Digestion of pRJ_00012 PCR Product - Fragment 2, Digestion 
                     of pRJ_00013 PCR Product - Fragment 2, pRJ_00009, pRJ_00009
                     PCR Product, pRJ_00010, pRJ_00010 PCR Product, pRJ_00011, 
                     pRJ_00011 PCR Product, pRJ_00012, pRJ_00012 PCR Product, 
                     pRJ_00013, pRJ_00013 PCR Product, pRJ_00095, pRJ_00096, 
                     pRJ_00097, pRJ_00098, pRJ_00099"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/created_by=User"
                     /note="/modified_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     primer_bind     2425..2445
                     /label=JH1711F
                     /note="color: black; sequence: 
                     ATGGGGTACCCATACGACGTGCCAGATTACGCCGGCCCCAAGAAGAAGAGGAAG"
     primer_bind     complement(2439..2457)
                     /label=RJ_00096Ra
                     /note="color: black; sequence: 
                     GGCGTAATCTGGCACGTCGTATGGGTAGCCAGATCCCACCTTCCTC"
     primer_bind     2449..2476
                     /label=RJ_00096Fe
                     /note="color: black; sequence: 
                     GTGCCAGATTACGCCGGATCAGGTCGGGATTCCAGGGAAGGGA"
     misc_feature    2449..2457
                     /label=GSG
                     /note="Geneious type: motif"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    2458..3027
                     /label=Rta
                     /note="Geneious type: domain"
                     /note="/Transferred_From=Digestion of pRJ_00010 PCR Product
                     - Fragment 2, Digestion of pRJ_00013 PCR Product - Fragment
                     2, pRJ_00010, pRJ_00010 PCR Product, pRJ_00013, pRJ_00013 
                     PCR Product, pRJ_00096, pRJ_00099"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/created_by=User"
                     /note="/modified_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     primer_bind     2533..2550
                     /label=FCS5
                     /note="color: black; sequence: GAGGTGTGCCAGCCAAAA"
     misc_feature    2545..2709
                     /label=Pro-rich
                     /note="Geneious type: Compositionally_biased"
                     /note="/Transferred_From=Rta (pBjh_4067)"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     primer_bind     complement(3005..3027)
                     /label=RJ_00050Rb
                     /note="color: black; sequence: 
                     CAGATGGTCTCTCGTCAAACAGAGATGTGTCGAAGATGG"
     misc_feature    3031..3058
                     /label=L0.3_Inert (concatenated sequence 6)
                     /note="Geneious type: Concatenated sequence"
                     /note="/Source"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3031..3034
                     /label=pL0 G-3
                     /note="Geneious type: gg_overhang"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3031..3034
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3031..3034
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3"
     misc_feature    3031..3034
                     /label=ligation
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3035..3054
                     /label=Inert
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3055..3111
                     /label=pBjh_4560 (concatenated sequence 7)
                     /note="Geneious type: Concatenated sequence"
                     /note="/Source"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3055..3058
                     /label=pL0 3-T
                     /note="Geneious type: gg_overhang"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3055..3058
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3"
     misc_feature    3055..3058
                     /label=ligation
                     /note="color: #a6acb3; direction: RIGHT"
     terminator      3059..3107
                     /label=Synth Terminator (Short)
                     /note="/ApEinfo_fwdcolor=cyan"
                     /note="/ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {}
                     0} width 5 offset 0"
                     /note="/ApEinfo_revcolor=green"
                     /note="/modified_by=User"
                     /note="color: #ffffff; direction: RIGHT"
     polyA_signal    3059..3064
                     /label=Poly-A Signal
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     polyA_signal    complement(3070..3075)
                     /label=Poly-A Signal
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: LEFT"
     misc_feature    3108..5172
                     /label=L1.Seq1-Seq2_Backbone (concatenated sequence 1)
                     /note="Geneious type: Concatenated sequence"
                     /note="/Source"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3108..3111
                     /label=pL0 T
                     /note="Geneious type: gg_overhang"
                     /note="/created_by=User"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3108..3111
                     /label=Ligation
                     /note="Geneious type: ligation"
                     /note="color: #a6acb3"
     misc_feature    3108..3111
                     /label=ligation
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3112..3156
                     /label=seq2
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    complement(3151..3156)
                     /label=BBb scar
                     /note="color: #a6acb3; direction: LEFT"
     misc_feature    3203..3312
                     /label=RK2 trfa
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    3317..3322
                     /label=BBb scar
                     /note="color: #a6acb3; direction: RIGHT"
     rep_origin      complement(3340..4022)
                     /direction=LEFT
                     /label=ColE1 origin
                     /note="color: #ffff00"
     misc_feature    complement(3343..3962)
                     /label=pBR322 origin
                     /note="color: #a6acb3; direction: LEFT"
     misc_feature    3938..3943
                     /label=BBb scar
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    complement(3949..3954)
                     /label=BBb scar
                     /note="color: #a6acb3; direction: LEFT"
ORIGIN
        1 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
       61 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
      121 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
      181 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagaac
      241 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
      301 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
      361 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
      421 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
      481 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
      541 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
      601 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
      661 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
      721 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
      781 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
      841 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
      901 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
      961 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     1021 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacgaattgg ccagcgctgc
     1081 attaccctgt tatccctaac cggtttaccg agctcttatt ggttttcaaa cttcattgac
     1141 tgtgccggag ttctgcaatt catgtgatct gcgcctggga gctcacgggg acagcccccc
     1201 cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc
     1261 ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga
     1321 cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc
     1381 tttgagcctg cagacacctg gggggatacg gggaaaaagc taggctgaaa gaggatctta
     1441 gaatgacacg cgcctgggag ctcacgggga cagccccccc ccaaagcccc cagggatgta
     1501 attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg
     1561 gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt
     1621 ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg
     1681 ggggatacgg ggaaaaagct ggatcataac tcgacgtgca tactgtcccc tccaccccac
     1741 agtggggaca acttgtgccg tatcagtccc ctccacccca cagtggggaa tgccatcagt
     1801 cgtttggtcc cctccacccc acagtggggt tttaccctag tctacaagtc ccctccaccc
     1861 cacagtgggg cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt
     1921 gaaccgtcag atcgcctgga gaattccaga taagtttgta caaaaaagag aggtgccacc
     1981 atgaagctac tgtcctctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag
     2041 tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac
     2101 tctcccaaaa ccaaaaggtc cccgctgact agggcacatc tgacagaagt ggaatcaagg
     2161 ctagaaagac tggaacagct atttctactg atttttcctc gagaggacct tgacatgatt
     2221 ttgaaaatgg attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat
     2281 aatgtgaata aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta
     2341 acattgagac agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt
     2401 caaagacagt tgactgtatc gatgggcccc aagaagaaga ggaaggtggg atctggccgg
     2461 gattccaggg aagggatgtt tttgccgaag cctgaggccg gctccgctat tagtgacgtg
     2521 tttgagggcc gcgaggtgtg ccagccaaaa cgaatccggc catttcatcc tccaggaagt
     2581 ccatgggcca accgcccact ccccgccagc ctcgcaccaa caccaaccgg tccagtacat
     2641 gagccagtcg ggtcactgac cccggcacca gtccctcagc cactggatcc agcgcccgca
     2701 gtgactcccg aggccagtca cctgttggag gatcccgatg aagagacgag ccaggctgtc
     2761 aaagcccttc gggagatggc cgatactgtg attccccaga aggaagaggc tgcaatctgt
     2821 ggccaaatgg acctttccca tccgccccca aggggccatc tggatgagct gacaaccaca
     2881 cttgagtcca tgaccgagga tctgaacctg gactcacccc tgaccccgga attgaacgag
     2941 attctggata ccttcctgaa cgacgagtgc ctcttgcatg ccatgcatat cagcacagga
     3001 ctgtccatct tcgacacatc tctgttttga gcttccactg gattgtacaa ttaccaacaa
     3061 taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgcgc tggtgcgttt
     3121 ttatgcttgt agtattgtat aatgttttta agatccttaa ttaataggga taacagggta
     3181 atatcttgcc ctgcacgaat accagcgacc ccttgcccaa atacttgccg tgggcctcgg
     3241 cctgagagcc aaaacacttg atgcggaaga agtcggtgcg ctcctgcttg tcgccggcat
     3301 cgttgcgcca catctaggat ctgccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3361 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3421 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3481 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3541 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3601 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3661 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3721 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3781 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3841 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3901 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3961 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4021 ga
//
