LOCUS       sequence-159168-        6653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RFP selectable marker for use in two-stage CRISPR-mediated allele 
            replacement strategies in Drosophila. D. americana tan homology arms
            can be replaced with locus of choice..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6653)
  AUTHORS   Lamb AM, Walker EA, Wittkopp PJ
  TITLE     Tools and strategies for scarless allele replacement in Drosophila 
            using CRISPR/Cas9.
  JOURNAL   Fly (Austin). 2016 Aug 5:1-12.
  PUBMED    27494619
REFERENCE   2  (bases 1 to 6653)
  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: "Fly 
            (Austin). 2016 Aug 5:1-12."
COMMENT     Sequence Label: pGEM-3XP3.RFP-tan
FEATURES             Location/Qualifiers
     source          1..6653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..88
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     80..97
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     81..97
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     104..123
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        104..122
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      153..162
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      157..166
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1028..1061
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3898..3916)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3899..3916)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3934..3950)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3934..3950)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3947..3969)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3958..3974
                     /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(3982..3988,3989..4006,4007..4012))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4027..4048
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4165..4182)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4336..4924)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4416..4435)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5095..5886,5887..5955))
                     /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     5718..5737
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5956..6060)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6138..6593)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6275..6296)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6487..6506
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
       61 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata
      121 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
      181 ctctggataa gcgtcagcct cgcgttgcgt cgcagtcgca gtcgcagtcg ccttcgctgc
      241 tggcgtcgca gcgtcgccca ttgctgttat tgttgttgct gtgtttctat atgcgttgcc
      301 tgctccgtta tcgcagcgac gtcgtcgtcg tcgtcgtcgt cgtcatcagc tgtcttcgtg
      361 ttgttgttgt gctttttgct gagtgttgtt tgttcggcac ctcgaattgc tgccgccaaa
      421 acatgtccaa ttcagtttga attctgcctt caagcgcttt agaacaatca tataaaaact
      481 tggcttgaat tatctatata caatttgttt acactgaaat tctctctctc tctcgtaacc
      541 tttctctcgc tctgtctctg tgtgtgcgtg cgtgtgtttg agtgttggta aagtttgttt
      601 aatgaaatta ttaactcagt tgtgtttttt tttattatta ttattatttc atcttgtgaa
      661 ttttgttttg tttttgttgc aaaatttgtt gtaattcact tttccagcga gtttttattc
      721 ccactgaatt attatatata tttcctgtgc tttatttttg ttttcgtctt tctatttaca
      781 ttttgtgatc gtggataaga cgaacccaag caaaaaaaaa aaaagaaaag aaaagaataa
      841 ttgagagcat attattactg ctcatcctgc tcgaatttca gagccaaata gcgcgaagag
      901 cgataaacgg tccacagcac atagaccaaa acgaaagccc aggaagccat gtgcacctcc
      961 ccggtggccc accaaatgtc gtccagcatg tcctccggca agatctcgta cggtaccaat
     1021 tgagctcata acttcgtata atgtatgcta tacgaagtta tgtcgacgaa ttcgcggccg
     1081 cgagctcgcc cggggatcta attcaattag agactaattc aattagagct aattcaatta
     1141 ggatccaagc ttatcgattt cgaaccctcg accgccggag tataaataga ggcgcttcgt
     1201 ctacggagcg acaattcaat tcaaacaagc aaagtgaaca cgtcgctaag cgaaagctaa
     1261 gcaaataaac aagcgcagct gaacaagcta aacaatcggg cggccgcact agagccggtc
     1321 gccaccatga ggtcttccaa gaatgttatc aaggagttca tgaggtttaa ggttcgcatg
     1381 gaaggaacgg tcaatgggca cgagtttgaa atagaaggcg aaggagaggg gaggccatac
     1441 gaaggccaca ataccgtaaa gcttaaggta accaaggggg gacctttgcc atttgcttgg
     1501 gatattttgt caccacaatt tcagtatgga agcaaggtat atgtcaagca ccctgccgac
     1561 ataccagact ataaaaagct gtcatttcct gaaggattta aatgggaaag ggtcatgaac
     1621 tttgaagacg gtggcgtcgt tactgtaacc caggattcca gtttgcagga tggctgtttc
     1681 atctacaagg tcaagttcat tggcgtgaac tttccttccg atggacctgt tatgcaaaag
     1741 aagacaatgg gctgggaagc cagcactgag cgtttgtatc ctcgtgatgg cgtgttgaaa
     1801 ggagagattc ataaggctct gaagctgaaa gacggtggtc attacctagt tgaattcaaa
     1861 agtatttaca tggcaaagaa gcctgtgcag ctaccagggt actactatgt tgactccaaa
     1921 ctggatataa caagccacaa cgaagactat acaatcgttg agcagtatga aagaaccgag
     1981 ggacgccacc atctgttcct ttagcggcca tcgaattcga gctcgcccac taagcgtcgc
     2041 gccacttcaa cgctcgatgg gagcgtcatt ggtgggcggg gtaaccgtcg aaatcagtgt
     2101 ttacgcttcc aatcgcaaca aaaaattcac tgcaacactg aaaagcatac gaaaacgatg
     2161 aagattgtac gagaaaccat aaagtatttt atccacaaag acacgtatag cagaaaagcc
     2221 aagttaactc ggcgataagt tgtgtacaca agaataaaat cggccagatt cagtgttgtc
     2281 agaaataaga aaaccccact atgtttttct ttgccttttc tttctcccag cgatcattca
     2341 tttcgtggtg aaagaacggg gtcattgcac ggagtttcga ctgcgggaaa gcagagctgc
     2401 cgttcacttc gtctataatt agcgctttct attttccccg attcgggccg ctgctgcgct
     2461 tttccgcctg ctgtttgtgg caagtgtagc agcaggctgt gcacgcagtg tggcatgcac
     2521 ttggctttcc accgttggta tcgattctct gggacgatga gtcattcctt tcggggccac
     2581 agcataatcg ttgccagctc accgaaatgg tgacttcatt tcttaactgc cgtcaagcat
     2641 gcgattgtac atacatacat atttatatat gtacatattt atgtgactat ggtaggtcga
     2701 tataatagca atcaacgcaa gcaaatgtgt cagtcctgct tacaggaacg attctattta
     2761 gtaattttcg ttgtataaag taattatgta tgtatgtaag ccccataaat ctgaaacaat
     2821 taggcaaaac catgcgaagc tctctggcgc ctaaggatcg atgctaggtc tgacttttat
     2881 atagaataat cttgccagat ttgcatcttt ctctgagtga actttgtttg attctctctc
     2941 tctcgctctc gccctcgctt tctcgttctc gctctcgctc tctctctctt atcagcaggt
     3001 gcaattacag ctacatatag taatcacata aagataatat gatcctgata agcgtttata
     3061 cgattcgact agtatatcta cttgtttctt tcttctactt ttttgccatt tcgtgaccca
     3121 gttccttaac ttaaccttca ctcaaaacta acactcactc acactcacac acacacacac
     3181 acacacacac aaatataaca atcactcact tgttcgcttt gagccggaaa aacaaaaaaa
     3241 gaaaaaaaaa gtggccaata tcaagggcaa caattggcat gcaggcccga ctccgtctgg
     3301 gcgagctgct ggtggggttg ggttggcagg tgagcgggcg ctgggccggg ttgcccgtgg
     3361 tctggaccca ctcctgctcc gtcgagcagt ccccgcgcac ataattaaca agctggcaac
     3421 aagatctatg tccaacaata aaccattatt cataatgttg tttttagcgt ttcgttttgt
     3481 ttgtttgccc tgtcgatcag tcgtgatcga ttatggttct ttttgttgtt gttgttgttg
     3541 ttgtatattt tgtaaaatat attttttgcc cagcagcagc atattagcat acaatacgtt
     3601 taaagagggc aatttgcaaa cggagttcga atcaaatatt tagaaagccg cttgcaattc
     3661 aattcaatat ttccaattaa tcataataat atacattata tatatatata tatataaata
     3721 tatgtgtgtg tgctttgcag ggtcgcactt ttggctcgat gatcaagaac tttctgatct
     3781 tatcgcagcc actgaacgag acctacttgc cgctctatgg caccgcaaaa ggcagtaagt
     3841 actactccac ggaccaggtt gcttctcaga tacgcgttgg atgcatagct tgagtattct
     3901 atagtgtcac ctaaatagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3961 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4021 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4081 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4141 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4201 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4261 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4321 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4381 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4441 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4501 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4561 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4621 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4681 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4741 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4801 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4861 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4921 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4981 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5041 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5101 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5161 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5221 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5281 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5341 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5401 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5461 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5521 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5581 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5641 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5701 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5761 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5821 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5881 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5941 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6001 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6061 cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga
     6121 gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     6181 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     6241 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     6301 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6361 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     6421 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     6481 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     6541 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg
     6601 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gct
//
