LOCUS       sequence-348665-        5824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of nuclear localized EBFP2 under Ae. aegypti 
            Polyubiquitin promoter (AAEL003888), EBFP2 version of 
            pSL1180-HR-PUbECFP (Addgene #47917).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5824)
  AUTHORS   Viswanatha R, Mameli E, Rodiger J, Merckaert P, Feitosa-Suntheimer 
            F, Colpitts TM, Mohr SE, Hu Y, Perrimon N
  TITLE     Bioinformatic and cell-based tools for pooled CRISPR knockout 
            screening in mosquitos.
  JOURNAL   Nat Commun. 2021 Nov 24;12(1):6825. doi: 10.1038/s41467-021-27129-3.
  PUBMED    34819517
REFERENCE   2  (bases 1 to 5824)
  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: "Nat 
            Commun."; date: "2021-11-24"; volume: "12(1):6825. doi"; pages: " 
            10.1038/s41467-021-27129-3"
COMMENT     Sequence Label: pSL1180-HR-PUbEBFP2-NLS
FEATURES             Location/Qualifiers
     source          1..5824
                     /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"
     polyA_signal    288..422
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(312..331)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     366..385
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(563..583)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(593..1303,1304..1306,1307..1309))
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     1243..1264
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2952..2969)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2952..2968)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2961..2983)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3181..3636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3268..3287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3478..3499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3653..3672)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3772..3794
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3832..3850)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3918..4022
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4023..4091,4092..4883)
                     /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(4241..4260)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5054..5642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5543..5562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5796..5813
                     /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 attcgaatgg
      241 ccatgggacg tcgacctgag gtaattataa cccgggccct atatatggat ccagacatga
      301 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
      361 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
      421 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
      481 tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcag ttatctagat
      541 ccggtggatc ttacgggtcc tccaccttcc gctttttctt gggtcgagat ctcttgtaca
      601 gctcgtccat gccgagagtg atcccggcgg cggtgcggaa ctccagcagg accatgtgat
      661 cgcgcttctc gttggggtct ttgctcagca cggactgggt gctcaggtag tggctgtcgg
      721 gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca
      781 cgctgccgtc ctccacgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct
      841 tgacggccat gatatagatg ttgtggctgt tgaagttgta ctccagcttg tgccccagga
      901 tgttgccgtc ctccttgaag tcgacgccct tcagctcgat gcggttcact agggtgtcgc
      961 cctcgaactt cacctcggcg cgggtcttgt aggtgccgtc gtccttgaag aagatggtgc
     1021 gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt
     1081 cggggtagcg ggcgaagcac tgcacgccgt ggctcagggt ggtcacgagg gtgggccagg
     1141 gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg ttggtggcat
     1201 cgccctcgcc ctcgcccctc acgctgaact tgtggccgtt tacgtcgccg tccagctcga
     1261 ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg ctagcgttga
     1321 aatctctgtt gagcagaaaa agaaacgagg aaacgcttga gtaattggtt gtgaaatgca
     1381 aactctcatt tgatattgat tcattgcctt tggcttcgag cacgacacga caggttttaa
     1441 acttgttttg ctttgtctgc gtttgcagtc gcaggccaag tgaaaaatat acacttgaag
     1501 gtgatgacgt cacaacaacg ccctactttt aagtgaaaaa ttaacttgtt ttcgactttg
     1561 aactacgtag ttttgaaatt gcgtatcttc aagttttcac gatttcctca aggtttttct
     1621 cgatatgtgt taatattacc ttaatgggta attaccatca aaatatttat tttagatatg
     1681 tgacggagca aatacgttat tcttattatt ctagaaattt aattcaatta gtagcgatga
     1741 ttcaacgaaa tatgattatc gctgtgaatc acaattgggt tttatcaatg atgatgaaac
     1801 tgcgttgcaa attttcacta atcactcaaa gctcaatagt cgccatcttg gaaaatagtt
     1861 tgctcattca aagacaaagg aatcattcac caaactagtt ttcgctcata gctataattt
     1921 catcaattaa tttacctacc ttcactagaa gattcccttc accgaaatgc actttcacca
     1981 atttaatagt aattgtcctt tgaataaagc tttgttcact ctgaaatttt ctcctctggc
     2041 taattggatc actctttttc actagagact tcacttcact tgcactggca ctgcttactt
     2101 gggccgcgta atgttcactc cactaggaaa cgtattcgat tgagctggtt tcgcctttgc
     2161 aggggcgttt tatagacact gtcgtagtgg tggttgtact tctagaaaat ttcagcaata
     2221 cattcataca tatacctctg ttcggttgga tggctctata tcgatcgaat atgggtacca
     2281 tccctgtgtc tgaatggaca gcaaaacgtg cttgtgtctg ttagtcgttc attctgtacc
     2341 ttgaacgatg cagttcaact tctggcaaag acgtcaatgt acctaccctt cgtgtatatg
     2401 gcatagagag gaagatgtgc gaatgccttt ttcgatagag aaaggatttc ctgatttgat
     2461 cgacaatttc ggtagctaat cttagcttcg aatgtaatct gataaccaaa tccagagaaa
     2521 aatcagtaat ttgggaattt cacttgaatc attctaattg catcatttgc tgtattagtg
     2581 cagtcagcaa gtgacgtcaa cccttctaaa tcgatatact tctgggaagc tttctttctt
     2641 gtctggctca gctggtgcca aggcaaatta taattggatt caatgcacaa gctacatgta
     2701 aagatagcta gcatgcgcaa atttaaagcg ctgatatcag cgctttaaat ttgcgcatgc
     2761 tagctatagt tctagaggta ccggttgtta acgttagccg gctacgtata ctccggaata
     2821 ttaataggcc taggatgcat atggcggccg cctgcagctg gcgccatcga tacgcgtacg
     2881 tcgcgaccgc ggacatgtac agagctcgag aagtactagt ggccacgtgg gccgtgcacc
     2941 ttaagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     3001 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     3061 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     3121 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     3181 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3241 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3301 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3361 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     3421 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3481 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     3541 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3601 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     3661 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     3721 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3781 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     3841 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     3901 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3961 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4021 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4081 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4141 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4201 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4261 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4321 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4381 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4441 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4501 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4561 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4621 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4681 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4741 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4801 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4861 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4921 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4981 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5041 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5101 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5161 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5221 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5281 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5341 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5401 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5461 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5521 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5581 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5641 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5701 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5761 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5821 aaga
//
