LOCUS       sequence-319776-        7782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Selectable homology donor template for Aag2 cell AGO1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7782)
  AUTHORS   Rozen-Gagnon K, Yi S, Jacobson E, Novack S, Rice CM
  TITLE     A selectable, plasmid-based system to generate CRISPR/Cas9 gene 
            edited and knock-in mosquito cell lines.
  JOURNAL   Sci Rep. 2021 Jan 12;11(1):736. doi: 10.1038/s41598-020-80436-5.
  PUBMED    33436886
REFERENCE   2  (bases 1 to 7782)
  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: "Sci Rep."; 
            date: "2021-01-12"; volume: "11(1):736. doi"; pages: " 
            10.1038/s41598-020-80436-5"
COMMENT     Sequence Label: pSL1180-HR-Aag2-loxP-PUb-RFP-loxP-3xFLAG-AGO1
FEATURES             Location/Qualifiers
     source          1..7782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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)."
     primer_bind     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    1378..1411
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2903..3598
                     /codon_start=1
                     /product="red fluorescent protein from Entacmaea 
                     quadricolor"
                     /label=TurboRFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     protein_bind    3606..3639
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4910..4927)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4910..4926)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4919..4941)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5139..5594
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5226..5245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5436..5457
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5611..5630)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5730..5752
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5790..5808)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5876..5980
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5981..6049,6050..6841)
                     /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     complement(6199..6218)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7012..7600
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7501..7520
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7754..7771
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga attcgacatg
      241 taggacatgt ggggtagttc cagaaatcca gataaacgat ggacatttga attatcccca
      301 aacctccatc atgttctgtt acttgatatc aactcaagga accccgcact aaaattgaat
      361 ttcatggcta ctatgataac cgtgttaaac cgttttctaa aagtttctgt cgcatgactc
      421 gttacaaatg acgttacaaa tctataattt ctataaatcc cttcaaaatt gactcttgga
      481 aatttatctg tattaaaatt atataattat tttaaaagct ggtaaatcac caacattttg
      541 acatccattt catccagaat cgttctattg tttatgcggt gaatgccgaa aaaaatatac
      601 cttcaatgat tcattgccga tgtttcaact gaactcaaac tcagaaatat gaaatttcac
      661 gcatttagat agatgactac tcttttttca gagttttagt tcacagcaaa gataaaaggt
      721 aattcaaatg agagctgctg tagaaagttt ctcgctcaat gtatgatctc gccttaaagc
      781 cattggtaac caagtgtgta gcttgttgtt actacgcaaa cgaatggatt tacacgttac
      841 ttaaaaatgc tacaatgaag attctctatc tcccagtggc gatagttata ttttggtcca
      901 ttcatttgct taaaaacaac gagctacaca tcaatggctt ttagatcgag atcacaattt
      961 atgcgagatt tcctcaagta atttattgcc aagaaatttc gtgaataata ataattataa
     1021 tgattattct gtttcatgag cgcatccatt ttggagcgtc cgtaaaatga aacaaaacgg
     1081 taaaccgcaa cctggctgtt tacttgcagc ggtttttcgg tacctaaact tgcgtacaag
     1141 tcgaactcgt ctaaaacata acgcctaccc aggctgctgg tagcgaaacg ttggggggcg
     1201 atgaagaaaa cttgtctcac tcgccatcat acgagagttt cgcgacgagt tcagcagacg
     1261 tgcgtgttta gcgcgacgac gaaaagtgca aaaattcggc cgtaattagt tcgcttccgt
     1321 ttttccgagt gcctccgatc gttagcggac cgttccgggt gtgatttaag cgtgaaaata
     1381 acttcgtata atgtatgcta tacgaagtta ttatctttac atgtagcttg tgcattgaat
     1441 ccaattataa tttgccttgg caccagctga gccagacaag aaagaaagct tcccagaagt
     1501 atatcgattt agaagggttg acgtcacttg ctgactgcac taatacagca aatgatgcaa
     1561 ttagaatgat tcaagtgaaa ttcccaaatt actgattttt ctctggattt ggttatcaga
     1621 ttacattcga agctaagatt agctaccgaa attgtcgatc aaatcaggaa atcctttctc
     1681 tatcgaaaaa ggcattcgca catcttcctc tctatgccat atacacgaag ggtaggtaca
     1741 ttgacgtctt tgccagaagt tgaactgcat cgttcaaggt acagaatgaa cgactaacag
     1801 acacaagcac gttttgctgt ccattcagac acagggatgg tacccatatt cgatcgatat
     1861 agagccatcc aaccgaacag aggtatatgt atgaatgtat tgctgaaatt ttctagaagt
     1921 acaaccacca ctacgacagt gtctataaaa cgcccctgca aaggcgaaac cagctcaatc
     1981 gaatacgttt cctagtggag tgaacattac gcggcccaag taagcagtgc cagtgcaagt
     2041 gaagtgaagt ctctagtgaa aaagagtgat ccaattagcc agaggagaaa atttcagagt
     2101 gaacaaagct ttattcaaag gacaattact attaaattgg tgaaagtgca tttcggtgaa
     2161 gggaatcttc tagtgaaggt aggtaaatta attgatgaaa ttatagctat gagcgaaaac
     2221 tagtttggtg aatgattcct ttgtctttga atgagcaaac tattttccaa gatggcgact
     2281 attgagcttt gagtgattag tgaaaatttg caacgcagtt tcatcatcat tgataaaacc
     2341 caattgtgat tcacagcgat aatcatattt cgttgaatca tcgctactaa ttgaattaaa
     2401 tttctagaat aataagaata acgtatttgc tccgtcacat atctaaaata aatattttga
     2461 tggtaattac ccattaaggt aatattaaca catatcgaga aaaaccttga ggaaatcgtg
     2521 aaaacttgaa gatacgcaat ttcaaaacta cgtagttcaa agtcgaaaac aagttaattt
     2581 ttcacttaaa agtagggcgt tgttgtgacg tcatcacctt caagtgtata tttttcactt
     2641 ggcctgcgac tgcaaacgca gacaaagcaa aacaagttta aaacctgtcg tgtcgtgctc
     2701 gaagccaaag gcaatgaatc aatatcaaat gagagtttgc atttcacaac caattactca
     2761 agcgtttcct cgtttctttt tctgctcaac agagatttca acaccaagct cgataagctt
     2821 gttcgaatct cgagtgcgcg cttccggagg tatacaccta ggcggtacca ctgcagtgaa
     2881 ttcggagctc taccgcggcc gcatgagcga gctgatcaag gagaacatgc acatgaagct
     2941 gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa
     3001 gccctacgag ggcacccaga ccatgaagat caaggtggtc gagggcggcc ctctcccctt
     3061 cgccttcgac atcctggcta ccagcttcat gtacggcagc aaagccttca tcaaccacac
     3121 ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagaat
     3181 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagct tccagaacgg
     3241 ctgcatcatc tacaacgtca agatcaacgg ggtgaacttc ccatccaacg gccctgtgat
     3301 gcagaagaaa acacgcggct gggaggccaa caccgagatg ctgtaccccg ctgacggcgg
     3361 cctgagaggc cacagccaga tggccctgaa gctcgtgggc gggggctacc tgcactgctc
     3421 cttcaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcttcca
     3481 cttcgtggac cacagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca
     3541 gcacgagatg gctgtggcca agtactgcga cctccctagc aaactggggc acagataatt
     3601 aattaataac ttcgtataat gtatgctata cgaagttata tggattacaa ggatcacgat
     3661 ggagattaca aggatcacga tatcgattac aaggatgatg ataagtatcc ggtgggtcaa
     3721 cgtaagtacc gtttccggac gtttgaaaaa agtgcaattc acaagagaag aaaaaaaaag
     3781 tgttgacgag gggaccttcc ttcctccctc tcgtctctct cgctgtgtaa atatttcttc
     3841 ttcccccatg tgtgtaattt tcctggcctg ctgctctgtt tggtgcttct ctggacgttt
     3901 ttgtgcgtgt gtgttggtgt gatggggggc ttttcctgtg tacggacgaa gaactttgtg
     3961 tcggtgtgcg tgtgagatga ggccctcgcc agaatgaaaa taaacctcat caacaatgaa
     4021 aagtgtcacc actttatgga ataaaaaacg tttttatgca gaagagcatg tgaattatgt
     4081 atcacttggc gatggaattg ggaacattga tgcttccgga ccgatggaat aatggaaaac
     4141 cagatgtttc tgcacgttct ttggcacctt ggcaatgtgt actaatcaac acctccctcg
     4201 aaaaatatac ttggtgatca gaatgtttgt gtttgtgaat agtaggtagt ttccgtgtgt
     4261 gggtgtgtgt ctttgctaat catcctgctc cagaaaaaaa ggaaaaccac caattttaat
     4321 catcattttc tgcggcttcg gagactgata agaggaatac aactgtttgc aaacagtgga
     4381 ttgaaaacaa tggatagaag ttttattggt tcacttcagt tctaaacagc ggatccatct
     4441 gaaaagttat atgaaattgt accccctttt cagtgctggg aaattattgc cttttgtgtc
     4501 gctctttgct cacaactaca tacggagatg cagacagagc tcgcattatg ttggtggtgc
     4561 tctgtcgctg gccatccgag aatcggaaac atcgtgcgga tggaacatgg aagaagagga
     4621 cgttgtaggg tttaccatta tgtatatgcg ttcaggcgac atcagacgag gtgggtcttc
     4681 tcgaaatgat gggtggttga ctgagtagca cctgctgttc atgtgcggaa atggaataac
     4741 gtggagatgc aatgttcttc aacgctagaa ggattcgggg agacgaaaag caaatccggt
     4801 ggcggccgcc tgcagctggc gccatcgata cgcgtacgtc gcgaccgcgg acatgtacag
     4861 agctcgagaa gtactagtgg ccacgtgggc cgtgcacctt aagcttggca ctggccgtcg
     4921 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     4981 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     5041 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     5101 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     5161 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5221 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5281 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5341 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5401 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5461 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc
     5521 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5581 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5641 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5701 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5761 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5821 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5881 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5941 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     6001 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6061 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     6121 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     6181 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6241 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6301 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6361 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6421 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6481 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6541 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6601 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6661 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6721 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6781 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6841 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6901 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6961 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     7021 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     7081 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     7141 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     7201 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7261 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7321 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7381 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7441 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7501 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7561 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7621 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7681 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7741 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa ga
//
