LOCUS       Exported                8962 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses tRNA-flanked gRNAs targeting hid, eve, and hedgehog.
ACCESSION   .
VERSION     .
KEYWORDS    OA-1045E
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8962)
  AUTHORS   .
  TITLE     Engineering reproductive isolation in D. melanogaster
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8962)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8962
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             join(316..387,789..1062,1137..1791,1853..2168,2372..2503,
                     2574..3188)
                     /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    complement(3963..3996)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        4055..4475
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     protein_bind    5583..5652
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(5871..5889)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5910..5926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5910..5926)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5923..5945)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5934..5950
                     /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(5958..5988)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6003..6024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6141..6158)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6312..6900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6392..6411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7071..7931)
                     /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     7694..7713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7932..8036)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8062..8517)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8199..8220)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8411..8430
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8718..8740
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8732..8749
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8733..8749
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8756..8775
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8756..8774
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8808..8826)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
ORIGIN
        1 tcaaaatgac attccactca aagtcagcgc tgtttgcctc cttctctgtc cacagaaata
       61 tcgccgtctc tttcgccgct gcgtccgcta tctctttcgc caccgtttgt agcgttacgt
      121 agcgtcaatg tccgccttca gttgcatttt gtcagcggtt tcgtgacgaa gctccaagcg
      181 gtttacgcca tcaattaaac acaaagtgct gtgccaaaac tcctctcgct tcttattttt
      241 gtttgttttt tgagtgattg gggtggtgat tggttttggg tgggtaagca ggggaaagtg
      301 tgaaaaatcc cggcaatggg ccaagaggat caggagctat taattcgcgg aggcagcaaa
      361 cacccatctg ccgagcatct gaacaatgtg agtagtacat gtgcatacat cttaagttca
      421 cttgatctat aggaactgcg attgcaacat caaattgtct gcggcgtgag aactgcgacc
      481 cacaaaaatc ccaaaccgca attgcacaaa caaatagtga cacgaaacag attattctgg
      541 tagctgttct cgctatataa gacaattttt gagatcatat catgatcaag acatctaaag
      601 gcattcattt tcgactatat tcttttttac aaaaaatata acaaccagat attttaagct
      661 gatcctagat gcacaaaaaa taaataaaag tataaaccta cttcgtagga tacttcgggg
      721 tactttttgt tcggggttag atgagcataa cgcttgtagt tgatatttga gatcccctat
      781 cattgcaggg tgacagcgga gcggcttcgc agagctgcat taaccagggc ttcgggcagg
      841 ccaaaaacta cggcacgctc cggccaccca gtccgccgga ggactccggt tcagggagcg
      901 gccaactagc cgagaacctc acctatgcct ggcacaatat ggacatcttt ggggcggtca
      961 atcagccggg ctccggatgg cggcagctgg tcaaccggac acgcggacta ttctgcaacg
     1021 agcgacacat accggcgccc aggaaacatt tgctcaagaa cggtgagttt ctattcgcag
     1081 tcggctgatc tgtgtgaaat cttaataaag ggtccaatta ccaatttgaa actcagtttg
     1141 cggcgtggcc tatccgggcg aacttttggc cgtgatgggc agttccggtg ccggaaagac
     1201 gaccctgctg aatgcccttg cctttcgatc gccgcagggc atccaagtat cgccatccgg
     1261 gatgcgactg ctcaatggcc aacctgtgga cgccaaggag atgcaggcca ggtgcgccta
     1321 tgtccagcag gatgacctct ttatcggctc cctaacggcc agggaacacc tgattttcca
     1381 agccatggtg cggatgccac gacatctgac ctatcggcag cgagtggccc gcgtggatca
     1441 ggtgatccag gagctttcgc tcagcaaatg tcagcacacg atcatcggtg tgcccggcag
     1501 ggtgaaaggt ctgtccggcg gagaaaggaa gcgtctggca ttcgcctccg aggctctaac
     1561 cgatccgccg cttctgatct gcgatgagcc cacctccgga ctggactcct ttaccgccca
     1621 cagcgtcgtc caggtgctga agaagctgtc gcagaagggc aagaccgtca tcctgaccat
     1681 tcatcagccg tcttccgagc tgtttgagct ctttgacaag atccttctga tggccgaggg
     1741 cagggtagct ttcttgggca ctcccagcga agccgtcgac ttcttttcct agtgagttcg
     1801 atgtgtttat taagggtatc tagtattaca taacatctca actcctatcc agcgtgggtg
     1861 cccagtgtcc taccaactac aatccggcgg acttttacgt acaggtgttg gccgttgtgc
     1921 ccggacggga gatcgagtcc cgtgatcgga tcgccaagat atgcgacaat tttgccatta
     1981 gcaaagtagc ccgggatatg gagcagttgt tggccaccaa aaatctggag aagccactgg
     2041 agcagccgga gaatgggtac acctacaagg ccacctggtt catgcagttc cgggcggtcc
     2101 tgtggcgatc ctggctgtcg gtgctcaagg aaccactcct cgtaaaagtg cgacttattc
     2161 agacaacggt gagtggttcc agtggaaaca aatgatataa cgcttacaat tcttggaaac
     2221 aaattcgcta gattttagat agaattgcct gattccacac ccttcttagt ttttttcaat
     2281 gagatgtata gtttatagtt ttgcagaaga taaataaatt tcatttaact cgcgaatatt
     2341 aatgagatgc gagtaacatt ttaatttgca gatggttgcc atcttgattg gcctcatctt
     2401 tttgggccaa caactcacgc aagtgggtgt gatgaatatc aacggagcca tcttcctctt
     2461 cctgaccaac atgacctttc aaaacgtctt tgccacgata aatgtaagtc atgtttagaa
     2521 tacatttgca tttcaataat ttactaactt tctaatgaat cgattcgatt taggtgttca
     2581 cctcagagct gccagttttt atgagggagg cccgaagtcg actttatcgc tgtgacacat
     2641 actttctggg caaaacgatt gccgaattgc cgctttttct cacagtgcca ctggtcttca
     2701 cggcgattgc ctatccgatg atcggactgc gggccggagt gctgcacttc ttcaactgcc
     2761 tggcgctggt cactctggtg gccaatgtgt caacgtcctt cggatatcta atatcctgcg
     2821 ccagctcctc gacctcgatg gcgctgtctg tgggtccgcc ggttatcata ccattcctgc
     2881 tctttggcgg cttcttcttg aactcgggct cggtgccagt atacctcaaa tggttgtcgt
     2941 acctctcatg gttccgttac gccaacgagg gtctgctgat taaccaatgg gcggacgtgg
     3001 agccgggcga aattagctgc acatcgtcga acaccacgtg ccccagttcg ggcaaggtca
     3061 tcctggagac gcttaacttc tccgccgccg atctgccgct ggactacgtg ggtctggcca
     3121 ttctcatcgt gagcttccgg gtgctcgcat atctggctct aagacttcgg gcccgacgca
     3181 aggagtagcc gacatatatc cgaaataact gcttgttttt tttttttacc attattacca
     3241 tcgtgtttac tgtttattgc cccctcaaaa agctaatgta attatatttg tgccaataaa
     3301 aacaagatat gacctataga atacaagtat ttccccttcg aacatcccca caagtagact
     3361 ttggatttgt cttctaacca aaagacttac acacctgcat accttacatc aaaaactcgt
     3421 ttatcgctac ataaaacacc gggatatatt ttttatatac atacttttca aatcgcgcgc
     3481 cctcttcata attcacctcc accacaccac gtttcgtagt tgctctttcg ctgtctccca
     3541 cccgctctcc gcaacacatt caccttttgt tcgacgacct tggagcgact gtcgttagtt
     3601 ccgcgcgatt cggttcgctc aaatggttcc gagtggttca tttcgtctca atagaaatta
     3661 gtaataaata tttgtatgta caatttattt gctccaatat atttgtatat atttccctca
     3721 cagctatatt tattctaatt taatattatg actttttaag gtaatttttt gtgacctgtt
     3781 cggagtgatt agcgttacaa tttgaactga aagtgacatc cagtgtttgt tccttgtgta
     3841 gatgcatctc aaaaaaatgg tgggcataat agtgttgttt atatatatca aaaataacaa
     3901 ctataataat aagaatacat ttaatttaga aaatgcttgg atttcactgg aactaggcta
     3961 gcataacttc gtataatgta tgctatacga agttatgcta gcggatccgg gaattgggaa
     4021 ttgggcaata tttaaatggc ggcgcgccga attctttttt gctcacctgt gattgctcct
     4081 actcaaatac aaaaacatca aattttctgt caataaagca tatttattta tatttatttt
     4141 acaggaaaga attcctttta aagtgtattt taacctataa tgaaaaacga ttaaaaaaaa
     4201 tacataaaat aattcgaaaa tttttgaata gcccaggttg ataaaaattc atttcatacg
     4261 ttttataact tatgccccta agtatttttt gaccatagtg tttcaattct acattaattt
     4321 tacagagtag aatgaaacgc cacctactca gccaagaggc gaaaaggtta gctcgccaag
     4381 cagagagggc gccagtgctc actacttttt ataattctca acttcttttt ccagactcag
     4441 ttcgtatata tagacctatt ttcaatttaa cgtcggttaa ttaagggctt tgagtgtgtg
     4501 tagacatcaa gcatcggtgg ttcagtggta gaatgctcgc ctgccacgcg ggcggcccgg
     4561 gttcgattcc cggccgatgc aatcgtgcgg tgctgagagg tttaagagct atgctggaaa
     4621 cagcatagca agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     4681 ggtgcgggct ttgagtgtgt gtagacatca agcatcggtg gttcagtggt agaatgctcg
     4741 cctgccacgc gggcggcccg ggttcgattc ccggccgatg cagcacatgc acgtgcatga
     4801 gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac
     4861 ttgaaaaagt ggcaccgagt cggtgcgggc tttgagtgtg tgtagacatc aagcatcggt
     4921 ggttcagtgg tagaatgctc gcctgccacg cgggcggccc gggttcgatt cccggccgat
     4981 gcattccact tcccttgcgc atagtttaag agctatgctg gaaacagcat agcaagttta
     5041 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg ggctttgagt
     5101 gtgtgtagac atcaagcatc ggtggttcag tggtagaatg ctcgcctgcc acgcgggcgg
     5161 cccgggttcg attcccggcc gatgcatatg ccactcgacg ttcgatgttt aagagctatg
     5221 ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca
     5281 ccgagtcggt gctttttttg cctacctgga gcctgagagt tgttcaataa aataaaaatg
     5341 tttcgttttt ttgctttcgc cagtatttat tatttttcat caatatgtat tcaatttggt
     5401 atgtatttag taattgtaat atatagacaa tggttttccg ttgacgtaca tacatctgac
     5461 gtgtgtttat ttagacataa tagttatgtt ttcacatctt tttaatgttc gcttaatgcg
     5521 tatgcatgcg gccgcaacgg tacctctaga gatccactag tgtcgacgat gtaggtcacg
     5581 gtctcgaagc cgcggtgcgg gtgccagggc gtgcccttgg gctccccggg cgcgtactcc
     5641 acctcaccca tctggtccat catgatgaac gggtcgaggt ggcggtagtt gatcccggcg
     5701 aacgcgcggc gcaccgggaa gccctcgccc tcgaaaccgc tgggcgcggt ggtcacggtg
     5761 agcacgggac gtgcgacggc gtcggcgggt gcggatacgc ggggcagcgt cagcgggttc
     5821 tcgacggtca cggcgggcat gtcgacacta gttctagcca gcttttgttc cctttagtga
     5881 gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5941 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     6001 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6061 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6121 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     6181 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     6241 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     6301 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     6361 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6421 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6481 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6541 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6601 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6661 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6721 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6781 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6841 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6901 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6961 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     7021 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     7081 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     7141 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     7201 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     7261 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     7321 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     7381 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     7441 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7501 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7561 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7621 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7681 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7741 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7801 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7861 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7921 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7981 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     8041 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     8101 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     8161 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     8221 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     8281 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     8341 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     8401 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     8461 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     8521 attcgccatt caggctgcgc wrcksktrrs massrcrccc gccgcgctta atgcgccgct
     8581 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     8641 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     8701 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata
     8761 cgactcacta tagggcgaat tgggtacgta ccgggcccct agtatgtatg taagttaata
     8821 aaacccattt ttgcggaaag tagataaaaa aaacattttt tttttttact gcactggata
     8881 tcattgaact tatctgatca gttttaaatt tacttcgatc caagggtatt tgatgtacca
     8941 ggttctttcg attacctctc ac
//
