LOCUS       sequence-343230-        5287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PUb-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 5287)
  TITLE     fluorescence marker cassettes for CRISPR knock-in in mosquitoes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5287)
  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            "Unpublished"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-DsRedSV40 GGGG AAGA
FEATURES             Location/Qualifiers
     source          1..5287
                     /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(2109..2111,2112..2114,2115..2789)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
                     HLFL"
     primer_bind     complement(2292..2312)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2510..2529
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2704..2727
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2910..3031
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2947..2966)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3001..3020
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3054..3070)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3055..3071)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3099..3117)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3138..3154)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3138..3154)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3151..3173)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3162..3178
                     /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(3186..3192,3193..3210,3211..3216))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3231..3252
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3369..3386)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3540..4128)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3620..3639)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4299..5090,5091..5159))
                     /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     4922..4941
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5160..5264)
                     /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 caacaatgat ggtgcgctcc tccaagaacg tcatcaagga gttcatgcgc ttcaaggtgc
     2161 gcatggaggg caccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     2221 cctacgaggg ccacaacacc gtgaagctga aggtgaccaa gggcggcccc ctgcccttcg
     2281 cctgggacat cctgtccccc cagttccagt acggctccaa ggtgtacgtg aagcaccccg
     2341 ccgacatccc cgactacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     2401 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggct
     2461 gcttcatcta caaggtgaag ttcatcggcg tgaacttccc ctccgacggc cccgtaatgc
     2521 agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc
     2581 tgaagggcga gatccacaag gccctgaagc tgaaggacgg cggccactac ctggtggagt
     2641 tcaagtccat ctacatggcc aagaagcccg tgcagctgcc cggctactac tacgtggact
     2701 ccaagctgga catcacctcc cacaacgagg actacaccat cgtggagcag tacgagcgca
     2761 ccgagggccg ccaccacctg ttcctgtagc ggccgcgact ctagatcata atcagccata
     2821 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     2881 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     2941 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3001 gtggtttgtc caaactcatc aatgtatctt aaagatgaga ccatcaagct tatcgatacc
     3061 gtcgacctcg agggggggcc cggtaccagc ttttgttccc tttagtgagg gttaatttcg
     3121 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3181 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3241 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3301 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3361 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3421 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3481 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3541 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3601 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3661 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3721 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3781 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3841 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3901 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3961 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4021 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4081 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4141 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4201 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4261 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4321 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4381 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4441 gatccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4501 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4561 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4621 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4681 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4741 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4801 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4861 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4921 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4981 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5041 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5101 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5161 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5221 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5281 gtgccac
//
