LOCUS       sequence-160463-        4575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Modified from pCFD4, with the vermillion marker and the attB site 
            removed.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4575)
  AUTHORS   Ge DT, Tipping C, Brodsky MH, Zamore PD
  TITLE     Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome
            Using white Co-conversion.
  JOURNAL   G3 (Bethesda). 2016 Aug 26. pii: g3.116.032557. doi: 
            10.1534/g3.116.032557.
  PUBMED    27543296
REFERENCE   2  (bases 1 to 4575)
  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: "G3 
            (Bethesda). 2016 Aug 26. pii: g3.116.032557. doi: 
            10.1534/g3.116.032557."
COMMENT     Sequence Label: pCFD4d
FEATURES             Location/Qualifiers
     source          1..4575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..803
                     /label=dU6-1 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-1 
                     snRNA (Port et al., 2014)"
     misc_RNA        822..897
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        898..1318
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        1337..1412
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1723..1743)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1723..1741)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1762..1778)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1762..1778)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1775..1797)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1786..1802
                     /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(1810..1816,1817..1834,1835..1840))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1855..1876
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1993..2010)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2164..2752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2244..2263)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2923..3714,3715..3783))
                     /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     3546..3565
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3784..3888)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3914..4369)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4051..4072)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4263..4282
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4496..4518
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4510..4527
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4511..4527
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4534..4553
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4534..4552
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gaattcattt tcaacgtcct cgatagtata gtggttagta tccccgcctg tcacgcggga
       61 gaccggggtt caattccccg tcggggagaa tctgtgattc tttttttttt tcttttactt
      121 tgttatataa acaatttttg ttttaattga atctaatttg ccattgcttt taggaatctc
      181 aggcatccag caagcgtttg tccgccgaat cgcccatcag tgaagaagat cctgtggcgg
      241 ctacgaaaat ctccccggcc atgtcggcct ccacctccag cgaaaaaccc atcagcgagc
      301 tggccacctc tgtgctgacc caccgctttc cagactccac ctcctcaccc ggcgaacatg
      361 gccttggacg aatgcagttg tcgatccgct acagcgccca gcgtcaaaaa ctagacgtga
      421 ccatacacaa aatccagaag ataccacttc gcgatcccag caatatcccc gatccgtatg
      481 ttaagctgta tctgttgcct ggacgcacca aggagtcgaa acgcaagacg agcgtgatca
      541 aggacaactg caaccccgtc tacgatgcat cctttgagta cctgatttcc attgccgaac
      601 tcaggcagac ggaactggag gtgacggtgt gcacccaaaa gggattccta tccggcggta
      661 gtcccatcat tggcatggta ggtacccgaa agcaacccct tagttacaga cacagcgcgt
      721 acgtccttcg catccttatg attcccaagt acatattctg caagagtaca gtatatatag
      781 gaaagatatc cgggtgaact tcggggtctt cgagaagacc tgttttagag ctagaaatag
      841 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      901 tttgctcacc tgtgattgct cctactcaaa tacaaaaaca tcaaattttc tgtcaataaa
      961 gcatatttat ttatatttat tttacaggaa agaattcctt ttaaagtgta ttttaaccta
     1021 taatgaaaaa cgattaaaaa aaatacataa aataattcga aaatttttga atagcccagg
     1081 ttgataaaaa ttcatttcat acgttttata acttatgccc ctaagtattt tttgaccata
     1141 gtgtttcaat tctacattaa ttttacagag tagaatgaaa cgccacctac tcagccaaga
     1201 ggcgaaaagg ttagctcgcc aagcagagag ggcgccagtg ctcactactt tttataattc
     1261 tcaacttctt tttccagact cagttcgtat atatagacct attttcaatt taacgtcggg
     1321 gtcttcgaga agacctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     1381 atcaacttga aaaagtggca ccgagtcggt gcttttttgc ctacctggag cctgagagtt
     1441 gttcaataaa ataaaaatgt ttcgtttttt tgctttcgcc agtatttatt atttttcatc
     1501 aatatgtatt caatttggta tgtatttagt aattgtaata tatagacaat ggttttccgt
     1561 tgacgtacat acatctgacg tgtgtttatt tagacataat agttatgttt tcacatcttt
     1621 ttaatgttcg cttaatgcgt atgcattcta gacaattgtg ctcggcaaca gtatatttgt
     1681 ggtgtgccaa ccaacaacct gcaggagctc cagcttttgt tccctttagt gagggttaat
     1741 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     1801 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     1861 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1921 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1981 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2041 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2101 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2161 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2221 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2281 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2341 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2401 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2461 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2521 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2581 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     2641 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2701 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2761 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     2821 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     2881 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     2941 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3001 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3061 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3121 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3181 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3241 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3301 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3361 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3421 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3481 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3541 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3601 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     3661 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     3721 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     3781 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     3841 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     3901 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     3961 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     4021 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     4081 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     4141 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     4201 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4261 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4321 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     4381 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     4441 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     4501 tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg
     4561 ggtacaagct gatct
//
