LOCUS       sequence-343225-        4199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3xP3-YFP fluorescence marker cassette for gene knock-in in 
            mosquitoes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4199)
  TITLE     fluorescence marker cassettes for CRISPR knock-in in mosquitoes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4199)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pKSB- 3xP3-YFPsv40 GGGG AAGA
FEATURES             Location/Qualifiers
     source          1..4199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     674..690
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     674..690
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      974..983
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(980..982,983..985,986..1699)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1025..1046)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1286..1305)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1633..1654
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1822..1943
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1859..1878)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1913..1932
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1966..1982)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1967..1983)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(2011..2029)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2050..2066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2050..2066)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2063..2085)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2074..2090
                     /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(2098..2104,2105..2122,2123..2128))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2143..2164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2281..2298)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2452..3040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2532..2551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3211..4002,4003..4071))
                     /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     3834..3853
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4072..4176)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctccaccg
      661 cggtggcggc cgctctagaa ctagtggatc ccccgggctg caggaattcg atggtctctg
      721 gggatctaat tcaattagag actaattcaa ttagagctaa ttcaattagg atccaagctt
      781 atcgatttcg aaccctcgac cgccggagta taaatagagg cgcttcgtct acggagcgac
      841 aattcaattc aaacaagcaa agtgaacacg tcgctaagcg aaagctaagc aaataaacaa
      901 gcgcagctga acaagctaaa caatcggggt accgctagag tcgacggtac cgcgggcccg
      961 ggatccaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1021 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1081 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1141 gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg
     1201 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1261 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1321 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1381 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1441 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1501 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1561 gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga
     1621 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1681 ggacgagctg tacaagtaaa gcggccgcga ctctagatca taatcagcca taccacattt
     1741 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     1801 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     1861 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     1921 tccaaactca tcaatgtatc ttaaagatga gaccatcaag cttatcgata ccgtcgacct
     1981 cgaggggggg cccggtacca gcttttgttc cctttagtga gggttaattt cgagcttggc
     2041 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     2101 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2161 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2221 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     2281 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2341 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2401 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2461 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2521 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2581 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2641 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2701 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2761 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2821 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2881 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2941 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3001 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3061 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3121 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3181 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3241 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3301 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagatccacg
     3361 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3421 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3481 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3541 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3601 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3661 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3721 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3781 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3841 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3901 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3961 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4021 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4081 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4141 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
