LOCUS       sequence-343240-        5324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PUb-GFP 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 5324)
  TITLE     fluorescence marker cassettes for CRISPR knock-in in mosquitoes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5324)
  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            "Unpublished"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- PUb-GFPsv40 GGGG AAGA
FEATURES             Location/Qualifiers
     source          1..5324
                     /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"
     CDS             join(2106..2108,2109..2111,2112..2825)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2106..2108,2109..2111,2112..2822)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2151..2172)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2412..2431)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2759..2780
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2947..3068
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2984..3003)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3038..3057
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3091..3107)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3092..3108)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3136..3154)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3175..3191)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3175..3191)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3188..3210)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3199..3215
                     /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(3223..3229,3230..3247,3248..3253))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3268..3289
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3406..3423)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3577..4165)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3657..3676)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4336..5127,5128..5196))
                     /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     4959..4978
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5197..5301)
                     /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 atggtctcag
      721 gggatcttta catgtagctt gtgcattgaa tccaattata atttgccttg gcaccagctg
      781 agccagacaa gaaagaaagc ttcccagaag tatatcgatt tagaagggtt gacgtcactt
      841 tgctgactgc actaatacag caaatgatac aattagaatg attcaagtga aattcccaaa
      901 ttactgcttt gtctctggat ttggttatca gattacattc gaagctaaga atagctaccg
      961 aaattgtcga tcaaatcagg aaatcctttc tctatcgaaa aaggcattcg cacatctttc
     1021 tctgtatgcc atatacacga atggtaggta cattgacgtc tttgccagaa gttgaactgc
     1081 atcgttcaag gtacagaatg aacgactaac agacacaagc acgttttgct gtccattcag
     1141 acacagggat ggtacccata gtcgatcgat ttagagccat ccaaccgaac agaggtatat
     1201 gtatgaatgg attgcagaaa ttttctagaa gtacaaccac cactacggca gtgtctataa
     1261 aacgcccctg caaaggcaaa accagctcaa tcgaatacgt ttcctagtgg agtgaacatt
     1321 acgcggtcca agtaagcagt gccagtgcaa gtgaagtgaa gtctctagtg aaaaagagtg
     1381 atccaattag ccagaggaga aaatttcaga gtgaacaaag ctttgttcaa aggacaatta
     1441 ctattaaatt tgtgaaagtg catttcggtg aagggaatct tctagtgaag gtaggtaaat
     1501 taaatgatga aattatagct atgagcgaaa actagtttgg tgaatgattc ctttgtcttt
     1561 gaatgagcaa actattttcc aagatggcga ctattgagct ttgagtgatt agtgaaaatt
     1621 tgcaacgcag tttcatcatc attgataaaa cccaattgtg attcacggcg ataatcatat
     1681 ttcgttgaat catcgctgct aattgaatta aatttctaga gcaagcgcga attcgccata
     1741 tttctaaaat taaatattgt ggtgataatt acccattaag gtaatattaa cacatatcga
     1801 gaaaaacctt gaggaaatcg tgaaaacttg aagatacgca atttccaaac tacgtagttc
     1861 aaagtcgaaa acaagttaat ttttcactaa aaagtagggc gttgttgtga cgtcatcacc
     1921 ttcaagtgta tatttttcac ttggcctgcg actgcaaacg cagacaaagc aaaacaagtt
     1981 taaaacctgt cgtgtcgtgc tcgaagccaa aggcaatgaa tcaatatcaa atgagagttt
     2041 gcatttcaca accaattact gaagcgtttc ctcgtttctt tttctgctca acagagattt
     2101 caacaatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2161 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2221 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2281 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2341 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2401 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2461 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2521 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2581 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2641 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2701 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2761 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2821 agtaaagcgg ccgcgactct agatcaaatc agccatacca catttgtaga ggttttactt
     2881 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2941 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3001 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3061 gtatcttaaa gatgagacca tcaagcttat cgataccgtc gacctcgagg gggggcccgg
     3121 taccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata
     3181 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3241 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3301 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3361 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3421 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3481 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3541 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3601 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3661 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3721 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3781 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3841 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3901 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3961 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4021 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4081 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4141 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4201 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4261 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4321 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4381 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4441 cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga
     4501 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4561 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4621 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4681 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4741 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4801 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4861 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4921 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4981 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5041 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5101 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5161 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5221 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5281 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccac
//
