LOCUS       sequence-343229-        5361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PUb-mTurquoise 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 5361)
  TITLE     fluorescence marker cassettes for CRISPR knock-in in mosquitoes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5361)
  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            "Unpublished"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-NLSmTurqSV40 GGGG AAGA
FEATURES             Location/Qualifiers
     source          1..5361
                     /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             2112..2132
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2139..2141,2142..2144,2145..2855)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2184..2205)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2445..2464)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2792..2813
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2984..3105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3021..3040)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3075..3094
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3128..3144)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3129..3145)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3173..3191)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3212..3228)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3212..3228)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3225..3247)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3236..3252
                     /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(3260..3266,3267..3284,3285..3290))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3305..3326
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3443..3460)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3614..4202)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3694..3713)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4373..5164,5165..5233))
                     /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     4996..5015
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5234..5338)
                     /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 gatgtcactt
      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 caacaatggc cccaaagaag aagcgtaagg tcggtggaat ggtgagcaag ggcgaggagc
     2161 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2221 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2281 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgtcctggg
     2341 gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2401 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2461 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2521 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacttta
     2581 gcgacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     2641 tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag cagaacaccc
     2701 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc
     2761 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2821 ccgggatcac tctcggcatg gacgagctgt acaagtccgg atgatagatc tgacgggtga
     2881 tcaaatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     2941 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     3001 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3061 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaagat gagaccatca
     3121 agcttatcga taccgtcgac ctcgaggggg ggcccggtac cagcttttgt tccctttagt
     3181 gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3241 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3301 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3361 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3421 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3481 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3541 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3601 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3661 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3721 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3781 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3841 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3901 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3961 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4021 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4081 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4141 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4201 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4261 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4321 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4381 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4441 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4501 caatgatacc gcgagatcca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4561 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4621 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4681 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4741 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4801 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4861 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4921 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4981 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5041 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5101 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5161 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5221 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5281 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5341 catttccccg aaaagtgcca c
//
