LOCUS       sequence-343227-        4202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3xP3-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 4202)
  TITLE     fluorescence marker cassettes for CRISPR knock-in in mosquitoes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4202)
  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            "Unpublished"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-mTurqSV40 GGGG AAGA
FEATURES             Location/Qualifiers
     source          1..4202
                     /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..1696)
                     /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(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    1825..1946
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1862..1881)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1916..1935
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1969..1985)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1970..1986)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(2014..2032)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2053..2069)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2053..2069)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2066..2088)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2077..2093
                     /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(2101..2107,2108..2125,2126..2131))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2146..2167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2284..2301)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2455..3043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2535..2554)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3214..4005,4006..4074))
                     /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     3837..3856
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4075..4179)
                     /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 cctgtcctgg ggcgtgcagt 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 caactacttt agcgacaacg tctatatcac
     1441 cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagga
     1501 cggcggcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1561 gctgctgccc gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga
     1621 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1681 ggacgagctg tacaagtccg gatgatagat ctgacgggtg atcaaatcag ccataccaca
     1741 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     1801 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     1861 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     1921 ttgtccaaac tcatcaatgt atcttaaaga tgagaccatc aagcttatcg ataccgtcga
     1981 cctcgagggg gggcccggta ccagcttttg ttccctttag tgagggttaa tttcgagctt
     2041 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     2101 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     2161 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2221 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     2281 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2341 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2401 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2461 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2521 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2581 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2641 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2701 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2761 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2821 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2881 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2941 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3001 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3061 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3121 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3181 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3241 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3301 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagatcc
     3361 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3421 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3481 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3541 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3601 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3661 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3721 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3781 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3841 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3901 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3961 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4021 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4081 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4141 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4201 ac
//
