LOCUS       sequence-362157-        5858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dsRed marked transgene for Crispr co-conversion with w+ gRNA and 
            drop in cloning sites for 2 more gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5858)
  TITLE     Drosophila Crispr co-conversion
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5858)
  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            "Unpublished"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: pALuWBB
FEATURES             Location/Qualifiers
     source          1..5858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(63..80)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(234..822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(314..333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(993..1784,1785..1853))
                     /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     1616..1635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1854..1958)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1985..2441
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2072..2091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2282..2303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2567..2589
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2581..2598
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2582..2598
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2608..2627
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2608..2626
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2806..2815
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2812..3489
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(2942..2960)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2992..3012)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3404..3427
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3610..3731
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3647..3666)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3701..3720
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    3738..3771
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3904..3973
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    4215..4600
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        4614..5034
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5151..5226
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        5324..5399
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        5497..5572
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(5651..5671)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5651..5669)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5690..5706)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5690..5706)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5703..5725)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5714..5730
                     /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(5738..5744,5745..5762,5763..5768))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5783..5804
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
       61 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      121 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      181 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      241 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      301 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      361 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      421 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      481 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      541 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      601 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      661 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      721 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      781 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      841 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      901 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      961 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1021 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1081 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagatcca
     1141 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1201 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1261 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1321 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1381 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1441 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1501 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1561 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1621 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1681 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1741 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1801 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1861 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1921 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1981 cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2041 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2101 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2161 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2221 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2281 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2341 ttataaggga ttttgccgat ttcggcctat tggttaaaaa aatgagctga tttaacaaaa
     2401 atttaacgcg aattttaaca aaatattaac gcttacaatt tccattcgcc attcaggctg
     2461 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     2521 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     2581 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtaca
     2641 agcttatcga tttcgaaccc tcgaccgccg gagtataaat agaggcgctt cgtctacgga
     2701 gcgacaattc aattcaaaca agcaaagtga acacgtcgct aagcgaaagc taagcaaata
     2761 aacaagcgca gctgaacaag ctaaacaatc ggctcgaagc cggtcgccac catggcctcc
     2821 tccgaggacg tcatcaagga gttcatgcgc ttcaaggtgc gcatggaggg ctccgtgaac
     2881 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     2941 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc
     3001 cagttccagt acggctccaa ggtgtacgtg aagcaccccg ccgacatccc cgactacaag
     3061 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     3121 gtggtgaccg tgacccagga ctcctccctc caggacggct ccttcatcta caaggtgaag
     3181 ttcatcggcg tgaacttccc ctccgacggc cccgtaatgc agaagaaaac tatgggctgg
     3241 gaggcgtcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccacaag
     3301 gccctgaagc tgaaggacgg cggccactac ctggtggagt tcaagtccat ctacatggcc
     3361 aagaagcccg tgcagctgcc cggctactac tacgtggact ccaagctgga catcacctcc
     3421 cacaacgagg actacaccat cgtggagcag tacgagcgcg ccgagggccg ccaccacctg
     3481 ttcctgtagg ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta
     3541 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     3601 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3661 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3721 aatgtatctt aaccggtata acttcgtata atgtatgcta tacgaagtta tagaagagca
     3781 ctagtaaaga tctccatgca taaggcgcgc ctaggccttc tgcagctcga ggctgcatcc
     3841 aacgcgttgg gagctctccg gatcaattcg gcttcaggta ccgtcgacga tgtaggtcac
     3901 agtctcgaag ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc
     3961 cacctcaccc atctggtcca tcatgatgaa cgggtcgagg tggcggtagt tgatcccggc
     4021 gaacgcgcgg cgcaccggga agccctcgcc ctcgaaaccg ctgggcgcgg tggtcacggt
     4081 gagcacggga cgtgcgacgg cgtcggctgg tgcggatacg cggggcagcg tcagcgggtt
     4141 ctcgacggtc acggcgggca tgtcgacaag ccgaattgat ccactagaag gcctaattcg
     4201 gtacactagt tggccacgta ataagtgtgc gttgaattta ttcgcaaaaa cattgcatat
     4261 tttcggcaaa gtaaaatttt gttgcatacc ttatcaaaaa ataagtgctg catacttttt
     4321 agagaaacca aataattttt tattgcatac ccgtttttaa taaaatacat tgcataccct
     4381 cttttaataa aaaatattgc atactttgac gaaacaaatt ttcgttgcat acccaataaa
     4441 agattattat attgcatacc cgtttttaat aaaatacatt gcataccctc ttttaataaa
     4501 gaatattgca tacgttgacg aaacaaattt tcgttgcata cccaataaaa gattattata
     4561 ttgcatacct tttcttgcca taccatttag ccgatcaatt gagatctgaa ttcttttttg
     4621 ctcacctgtg attgctccta ctcaaataca aaaacatcaa attttctgtc aataaagcat
     4681 atttatttat atttatttta caggaaagaa ttccttttaa agtgtatttt aacctataat
     4741 gaaaaacgat taaaaaaaat acataaaata attcgaaaat ttttgaatag cccaggttga
     4801 taaaaattca tttcatacgt tttataactt atgcccctaa gtattttttg accatagtgt
     4861 ttcaattcta cattaatttt acagagtaga atgaaacgcc acctactcag ccaagaggcg
     4921 aaaaggttag ctcgccaagc agagagggcg ccagtgctca ctacttttta taattctcaa
     4981 cttctttttc cagactcagt tcgtatatat agacctattt tcaatttaac gtcggggctt
     5041 tgagtgtgtg tagacatcaa gcatcggtgg ttcagtggta gaatgctcgc ctgccacgcg
     5101 ggcggcccgg gttcgattcc cggccgatgc ataccattcc tgctctttgg gttttagagc
     5161 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     5221 cggtgctaac aaagcaccag tggtctagtg gtagaatagt accctgccac ggtacagacc
     5281 cgggatcctt cgattcccgg ctggtgcaga gaccagtggt ctcgttttag agctagaaat
     5341 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     5401 aacaaagcac cagtggtcta gtggtagaat agtaccctgc cacggtacag acccggcttc
     5461 gattcacggc tggtgcaggt cttcgtcgac gaagacgttt tagagctaga aatagcaagt
     5521 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctctctaga
     5581 caattgtgct cggcaacagt atatttgtgg tgtgccaacc aacaacctgc aggagctcca
     5641 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     5701 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5761 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5821 tgcccgcttt ccagtcggga aacctgtcgt gccagctg
//
