LOCUS       sequence-342920-        4158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3xP3-DsRed 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 4158)
  TITLE     fluorescence marker cassettes for CRISPR knock-in in mosquitoes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4158)
  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            "Unpublished"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-DsRedSV40 GGGG AAGA
FEATURES             Location/Qualifiers
     source          1..4158
                     /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..1660)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
                     HLFL"
     primer_bind     complement(1163..1183)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1381..1400
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1575..1598
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1781..1902
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1818..1837)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1872..1891
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1925..1941)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1926..1942)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(1970..1988)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2009..2025)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2009..2025)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2022..2044)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2033..2049
                     /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(2057..2063,2064..2081,2082..2087))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2102..2123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2240..2257)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2411..2999)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2491..2510)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3170..3961,3962..4030))
                     /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     3793..3812
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4031..4135)
                     /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 tggtgcgctc ctccaagaac gtcatcaagg agttcatgcg
     1021 cttcaaggtg cgcatggagg gcaccgtgaa cggccacgag ttcgagatcg agggcgaggg
     1081 cgagggccgc ccctacgagg gccacaacac cgtgaagctg aaggtgacca agggcggccc
     1141 cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca aggtgtacgt
     1201 gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg
     1261 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     1321 gcaggacggc tgcttcatct acaaggtgaa gttcatcggc gtgaacttcc cctccgacgg
     1381 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgcc tgtacccccg
     1441 cgacggcgtg ctgaagggcg agatccacaa ggccctgaag ctgaaggacg gcggccacta
     1501 cctggtggag ttcaagtcca tctacatggc caagaagccc gtgcagctgc ccggctacta
     1561 ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacacca tcgtggagca
     1621 gtacgagcgc accgagggcc gccaccacct gttcctgtag cggccgcgac tctagatcat
     1681 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     1741 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     1801 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     1861 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaagatgag accatcaagc
     1921 ttatcgatac cgtcgacctc gagggggggc ccggtaccag cttttgttcc ctttagtgag
     1981 ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2041 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2101 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2161 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2221 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2281 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2341 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2401 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2461 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2521 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2581 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2641 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2701 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2761 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2821 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     2881 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2941 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3001 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3061 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3121 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3181 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3241 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3301 tgataccgcg agatccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3361 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3421 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3481 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3541 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3601 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3661 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3721 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3781 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3841 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3901 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3961 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4021 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4081 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4141 ttccccgaaa agtgccac
//
