LOCUS       sequence-343241-        5254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PUb-GFP fluorescence marker cassette for gene knock-in in 
            mosquitoes, with DmTub56D terminator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5254)
  TITLE     fluorescence marker cassettes for CRISPR knock-in in mosquitoes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5254)
  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            "Unpublished"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-GFPTub56D GGGG AAGA
FEATURES             Location/Qualifiers
     source          1..5254
                     /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(2101..2103,2104..2106,2107..2820)
                     /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(2101..2103,2104..2106,2107..2817)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2146..2167)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2407..2426)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2754..2775
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3021..3037)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3022..3038)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3066..3084)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3105..3121)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3105..3121)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3118..3140)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3129..3145
                     /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(3153..3159,3160..3177,3178..3183))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3198..3219
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3336..3353)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3507..4095)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3587..3606)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4266..5057,5058..5126))
                     /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     4889..4908
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5127..5231)
                     /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 atggtctcat
      721 ctttacatgt agcttgtgca ttgaatccaa ttataatttg ccttggcacc agctgagcca
      781 gacaagaaag aaagcttccc agaagtatat cgatttagaa gggttgacgt aactttgctg
      841 actgcactaa tacagcaaat gatacaatta gaatgattca agtgaaattc ccaaattact
      901 gctttgtctc tggatttggt tatcagatta cattcgaagc taagaatagc taccgaaatt
      961 gtcgatcaaa tcaggaaatc ctttctctat cgaaaaaggc attcgcacat ctttctctgt
     1021 atgccatata cacgaatggt aggtacattg acgtctttgc cagaagttga actgcatcgt
     1081 tcaaggtaca gaatgaacga ctaacagaca caagcacgtt ttgctgtcca ttcagacaca
     1141 gggatggtac ccatagtcga tcgatttaga gccatccaac cgaacagagg tatatgtatg
     1201 aatggattgc agaaattttc tagaagtaca accaccacta cggcagtgtc tataaaacgc
     1261 ccctgcaaag gcaaaaccag ctcaatcgaa tacgtttcct agtggagtga acattacgcg
     1321 gtccaagtaa gcagtgccag tgcaagtgaa gtgaagtctc tagtgaaaaa gagtgatcca
     1381 attagccaga ggagaaaatt tcagagtgaa caaagctttg ttcaaaggac aattactatt
     1441 aaatttgtga aagtgcattt cggtgaaggg aatcttctag tgaaggtagg taaattaaat
     1501 gatgaaatta tagctatgag cgaaaactag tttggtgaat gattcctttg tctttgaatg
     1561 agcaaactat tttccaagat ggcgactatt gagctttgag tgattagtga aaatttgcaa
     1621 cgcagtttca tcatcattga taaaacccaa ttgtgattca cggcgataat catatttcgt
     1681 tgaatcatcg ctgctaattg aattaaattt ctagagcaag cgcgaattcg ccatatttct
     1741 aaaattaaat attgtggtga taattaccca ttaaggtaat attaacacat atcgagaaaa
     1801 accttgagga aatcgtgaaa acttgaagat acgcaatttc caaactacgt agttcaaagt
     1861 cgaaaacaag ttaatttttc actaaaaagt agggcgttgt tgtgacgtca tcaccttcaa
     1921 gtgtatattt ttcacttggc ctgcgactgc aaacgcagac aaagcaaaac aagtttaaaa
     1981 cctgtcgtgt cgtgctcgaa gccaaaggca atgaatcaat atcaaatgag agtttgcatt
     2041 tcacaaccaa ttactgaagc gtttcctcgt ttctttttct gctaaacaga gatttcaacc
     2101 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2161 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2221 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2281 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2341 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2401 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2461 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2521 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2581 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2641 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2701 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2761 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     2821 attcgaatcg gaaatcaatc gaattctcga tcaatgtttt gcgaaaaaaa atgaaacgag
     2881 aacaaatgaa ttcagttgta aaagatttgt tctcttaaca acgtaatcaa taaagtcgga
     2941 tccaattttg ttcacaagtc taaacaatta tggtttttgt tggggtttcg tataggaacc
     3001 cctgagacca tcaagcttat cgataccgtc gacctcgagg gggggcccgg taccagcttt
     3061 tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct
     3121 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3181 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3241 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3301 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3361 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3421 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3481 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3541 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3601 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3661 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3721 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3781 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3841 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3901 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3961 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4021 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4081 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4141 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4201 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4261 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4321 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4381 ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga tttatcagca
     4441 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4501 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4561 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4621 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     4681 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     4741 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     4801 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4861 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4921 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4981 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5041 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5101 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5161 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5221 ggggttccgc gcacatttcc ccgaaaagtg ccac
//
