LOCUS       sequence-314889-        7931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid supports expression of gRNA targeting Pol-gamma-35 site #2, 
            tagged with mini-White, and can be integrated with phC31..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7931)
  AUTHORS   Kandul NP, Liu J, Bennett JB, Marshall JM, Akbari OS
  TITLE     A confinable home and rescue gene drive for population modification.
  JOURNAL   Elife. 2021 Mar 5;10. pii: 65939. doi: 10.7554/eLife.65939.
  PUBMED    33666174
REFERENCE   2  (bases 1 to 7931)
  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: "Elife."; 
            date: "2021-03-5"; volume: "10. pii: 65939. doi"; pages: " 
            10.7554/eLife.65939"
COMMENT     Sequence Label: 1026M=U6-3-gRNA#2[Pol-gamma-35]
FEATURES             Location/Qualifiers
     source          1..7931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..421
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        442..517
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    572..641
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(860..878)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(899..915)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(899..915)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(912..934)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    923..939
                     /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(947..953,954..971,972..977))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    992..1013
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1130..1147)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1301..1889)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1381..1400)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2060..2851,2852..2920))
                     /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     2683..2702
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2921..3025)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3051..3506)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3188..3209)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3400..3419
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3633..3655
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3647..3664
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3648..3664
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3671..3690
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3671..3689
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3723..3741)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(4193..4264,4666..4939,5014..5668,5730..6045,6249..6380,
                     6451..7065)
                     /codon_start=1
                     /gene="white"
                     /product="Drosophila white gene eye color pigment"
                     /label=mini-white
                     /note="This is a modified version of the white gene lacking
                     part of the first intron."
                     /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
                     GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
                     IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
                     QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
                     SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
                     VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
                     TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
                     ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
                     VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
                     FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
                     GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
                     NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
     protein_bind    7840..7873
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ttttttgctc acctgtgatt gctcctactc aaatacaaaa acatcaaatt ttctgtcaat
       61 aaagcatatt tatttatatt tattttacag gaaagaattc cttttaaagt gtattttaac
      121 ctataatgaa aaacgattaa aaaaaataca taaaataatt cgaaaatttt tgaatagccc
      181 aggttgataa aaattcattt catacgtttt ataacttatg cccctaagta ttttttgacc
      241 atagtgtttc aattctacat taattttaca gagtagaatg aaacgccacc tactcagcca
      301 agaggcgaaa aggttagctc gccaagcaga gagggcgcca gtgctcacta ctttttataa
      361 ttctcaactt ctttttccag actcagttcg tatatataga cctattttca atttaacgtc
      421 ggactgatgc agttgcgcag cgttttagag ctagaaatag caagttaaaa taaggctagt
      481 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttttttggt acctctagag
      541 atccactagt gtcgacgatg taggtcacgg tctcgaagcc gcggtgcggg tgccagggcg
      601 tgcccttggg ctccccgggc gcgtactcca cctcacccat ctggtccatc atgatgaacg
      661 ggtcgaggtg gcggtagttg atcccggcga acgcgcggcg caccgggaag ccctcgccct
      721 cgaaaccgct gggcgcggtg gtcacggtga gcacgggacg tgcgacggcg tcggcgggtg
      781 cggatacgcg gggcagcgtc agcgggttct cgacggtcac ggcgggcatg tcgacactag
      841 ttctagccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg taatcatggt
      901 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
      961 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     1021 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1081 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     1141 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1201 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     1261 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     1321 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     1381 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1441 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1501 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1561 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     1621 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     1681 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     1741 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     1801 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     1861 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     1921 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     1981 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     2041 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2101 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2161 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     2221 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2281 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2341 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     2401 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     2461 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     2521 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     2581 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     2641 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     2701 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     2761 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     2821 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     2881 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     2941 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3001 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     3061 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     3121 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     3181 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     3241 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     3301 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     3361 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     3421 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     3481 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     3541 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     3601 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     3661 cagtgaattg taatacgact cactataggg cgaattgggt acgtaccggg cccctagtat
     3721 gtatgtaagt taataaaacc catttttgcg gaaagtagat aaaaaaaaca tttttttttt
     3781 ttactgcact ggatatcatt gaacttatct gatcagtttt aaatttactt cgatccaagg
     3841 gtatttgatg taccaggttc tttcgattac ctctcactca aaatgacatt ccactcaaag
     3901 tcagcgctgt ttgcctcctt ctctgtccac agaaatatcg ccgtctcttt cgccgctgcg
     3961 tccgctatct ctttcgccac cgtttgtagc gttacgtagc gtcaatgtcc gccttcagtt
     4021 gcattttgtc agcggtttcg tgacgaagct ccaagcggtt tacgccatca attaaacaca
     4081 aagtgctgtg ccaaaactcc tctcgcttct tatttttgtt tgttttttga gtgattgggg
     4141 tggtgattgg ttttgggtgg gtaagcaggg gaaagtgtga aaaatcccgg caatgggcca
     4201 agaggatcag gagctattaa ttcgcggagg cagcaaacac ccatctgccg agcatctgaa
     4261 caatgtgagt agtacatgtg catacatctt aagttcactt gatctatagg aactgcgatt
     4321 gcaacatcaa attgtctgcg gcgtgagaac tgcgacccac aaaaatccca aaccgcaatt
     4381 gcacaaacaa atagtgacac gaaacagatt attctggtag ctgttctcgc tatataagac
     4441 aatttttgag atcatatcat gatcaagaca tctaaaggca ttcattttcg actatattct
     4501 tttttacaaa aaatataaca accagatatt ttaagctgat cctagatgca caaaaaataa
     4561 ataaaagtat aaacctactt cgtaggatac ttcggggtac tttttgttcg gggttagatg
     4621 agcataacgc ttgtagttga tatttgagat cccctatcat tgcagggtga cagcggagcg
     4681 gcttcgcaga gctgcattaa ccagggcttc gggcaggcca aaaactacgg cacgctccgg
     4741 ccacccagtc cgccggagga ctccggttca gggagcggcc aactagccga gaacctcacc
     4801 tatgcctggc acaatatgga catctttggg gcggtcaatc agccgggctc cggatggcgg
     4861 cagctggtca accggacacg cggactattc tgcaacgagc gacacatacc ggcgcccagg
     4921 aaacatttgc tcaagaacgg tgagtttcta ttcgcagtcg gctgatctgt gtgaaatctt
     4981 aataaagggt ccaattacca atttgaaact cagtttgcgg cgtggcctat ccgggcgaac
     5041 ttttggccgt gatgggcagt tccggtgccg gaaagacgac cctgctgaat gcccttgcct
     5101 ttcgatcgcc gcagggcatc caagtatcgc catccgggat gcgactgctc aatggccaac
     5161 ctgtggacgc caaggagatg caggccaggt gcgcctatgt ccagcaggat gacctcttta
     5221 tcggctccct aacggccagg gaacacctga ttttccaagc catggtgcgg atgccacgac
     5281 atctgaccta tcggcagcga gtggcccgcg tggatcaggt gatccaggag ctttcgctca
     5341 gcaaatgtca gcacacgatc atcggtgtgc ccggcagggt gaaaggtctg tccggcggag
     5401 aaaggaagcg tctggcattc gcctccgagg ctctaaccga tccgccgctt ctgatctgcg
     5461 atgagcccac ctccggactg gactccttta ccgcccacag cgtcgtccag gtgctgaaga
     5521 agctgtcgca gaagggcaag accgtcatcc tgaccattca tcagccgtct tccgagctgt
     5581 ttgagctctt tgacaagatc cttctgatgg ccgagggcag ggtagctttc ttgggcactc
     5641 ccagcgaagc cgtcgacttc ttttcctagt gagttcgatg tgtttattaa gggtatctag
     5701 tattacataa catctcaact cctatccagc gtgggtgccc agtgtcctac caactacaat
     5761 ccggcggact tttacgtaca ggtgttggcc gttgtgcccg gacgggagat cgagtcccgt
     5821 gatcggatcg ccaagatatg cgacaatttt gccattagca aagtagcccg ggatatggag
     5881 cagttgttgg ccaccaaaaa tctggagaag ccactggagc agccggagaa tgggtacacc
     5941 tacaaggcca cctggttcat gcagttccgg gcggtcctgt ggcgatcctg gctgtcggtg
     6001 ctcaaggaac cactcctcgt aaaagtgcga cttattcaga caacggtgag tggttccagt
     6061 ggaaacaaat gatataacgc ttacaattct tggaaacaaa ttcgctagat tttagataga
     6121 attgcctgat tccacaccct tcttagtttt tttcaatgag atgtatagtt tatagttttg
     6181 cagaagataa ataaatttca tttaactcgc gaatattaat gagatgcgag taacatttta
     6241 atttgcagat ggttgccatc ttgattggcc tcatcttttt gggccaacaa ctcacgcaag
     6301 tgggtgtgat gaatatcaac ggagccatct tcctcttcct gaccaacatg acctttcaaa
     6361 acgtctttgc cacgataaat gtaagtcatg tttagaatac atttgcattt caataattta
     6421 ctaactttct aatgaatcga ttcgatttag gtgttcacct cagagctgcc agtttttatg
     6481 agggaggccc gaagtcgact ttatcgctgt gacacatact ttctgggcaa aacgattgcc
     6541 gaattgccgc tttttctcac agtgccactg gtcttcacgg cgattgccta tccgatgatc
     6601 ggactgcggg ccggagtgct gcacttcttc aactgcctgg cgctggtcac tctggtggcc
     6661 aatgtgtcaa cgtccttcgg atatctaata tcctgcgcca gctcctcgac ctcgatggcg
     6721 ctgtctgtgg gtccgccggt tatcatacca ttcctgctct ttggcggctt cttcttgaac
     6781 tcgggctcgg tgccagtata cctcaaatgg ttgtcgtacc tctcatggtt ccgttacgcc
     6841 aacgagggtc tgctgattaa ccaatgggcg gacgtggagc cgggcgaaat tagctgcaca
     6901 tcgtcgaaca ccacgtgccc cagttcgggc aaggtcatcc tggagacgct taacttctcc
     6961 gccgccgatc tgccgctgga ctacgtgggt ctggccattc tcatcgtgag cttccgggtg
     7021 ctcgcatatc tggctctaag acttcgggcc cgacgcaagg agtagccgac atatatccga
     7081 aataactgct tgtttttttt ttttaccatt attaccatcg tgtttactgt ttattgcccc
     7141 ctcaaaaagc taatgtaatt atatttgtgc caataaaaac aagatatgac ctatagaata
     7201 caagtatttc cccttcgaac atccccacaa gtagactttg gatttgtctt ctaaccaaaa
     7261 gacttacaca cctgcatacc ttacatcaaa aactcgttta tcgctacata aaacaccggg
     7321 atatattttt tatatacata cttttcaaat cgcgcgccct cttcataatt cacctccacc
     7381 acaccacgtt tcgtagttgc tctttcgctg tctcccaccc gctctccgca acacattcac
     7441 cttttgttcg acgaccttgg agcgactgtc gttagttccg cgcgattcgg ttcgctcaaa
     7501 tggttccgag tggttcattt cgtctcaata gaaattagta ataaatattt gtatgtacaa
     7561 tttatttgct ccaatatatt tgtatatatt tccctcacag ctatatttat tctaatttaa
     7621 tattatgact ttttaaggta attttttgtg acctgttcgg agtgattagc gttacaattt
     7681 gaactgaaag tgacatccag tgtttgttcc ttgtgtagat gcatctcaaa aaaatggtgg
     7741 gcataatagt gttgtttata tatatcaaaa ataacaacta taataataag aatacattta
     7801 atttagaaaa tgcttggatt tcactggaac taggctagca taacttcgta taatgtatgc
     7861 tatacgaagt tatgctagcg gatccgggaa ttgggaattg ggcaatattt aaatggcggc
     7921 gcgccgaatt c
//
