LOCUS       sequence-159157-        7290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP 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 7290)
  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 7290)
  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-WingGFP-tan
FEATURES             Location/Qualifiers
     source          1..7290
                     /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"
     regulatory      2324..2333
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2330..2332,2333..2335,2336..3046)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2330..2332,2333..2335,2336..3046)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2375..2396)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2636..2655)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2983..3004
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3298..3419
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3335..3354)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3389..3408
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(4535..4553)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4536..4553)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4571..4587)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4571..4587)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4584..4606)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4595..4611
                     /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(4619..4625,4626..4643,4644..4649))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4664..4685
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4802..4819)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4973..5561)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5053..5072)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5732..6523,6524..6592))
                     /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     6355..6374
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6593..6697)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6775..7230)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6912..6933)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7124..7143
                     /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 cggtaccatc
     1021 catactcatc aattatctta tagcttatcg ttacgtgtac ggcgcgccaa tggcgaaact
     1081 ggaaacacta aactagtaaa tgcgaaatgt atcatatgga ttttcatatt ttttccactg
     1141 ctaatggaaa tgtttgcctt gaagagattg gtcgactatt aaatgattat cgcccgatta
     1201 ccacattgag tggtttaaaa tagccataaa atatgcaact gacgatggct taagataaat
     1261 acgtcgcaga gtcactcata aatttcgaac gcagcccgct gatttaccta cccctctaaa
     1321 cgattcatag tatatgtacg agtatatcca ctaagctttt tcgagcactg attttttcgc
     1381 ttgcacgaga caagtgcacc accgcaattg caggcaaatt atgtctgagg taatgattcc
     1441 gtttcgtgca agattacaca gaaatcaaat tacgacaacc tttattcagt aagcaaacaa
     1501 agcctttgtt ggcatctaat tattccactt atggttgcga tttcgggagc tacaatcggt
     1561 tttggtttag tatatctagc gagttccttg gcgacattta aaatttacaa ataaagtttc
     1621 tctattcaat cgggacagtg gaaattgact attttattta tattaatgaa cttattttta
     1681 atttggctta agttactaag gggtactaat agtttgagcg cagtgcatgt catggggaca
     1741 tgtgcaattg tgtgtaagcg ggaagtgatc gcggccttcc gaatttggcc atgccaaata
     1801 atcccagctc gaaaggaggg gacccggcgg tcagggccat ggacattgaa cttgaaaaaa
     1861 aaaaaacaca aaaatatata acacaaaacg gaaaatgctg tgtaccgctt atgttagaga
     1921 agttgagcaa cgggtttttc gttttgcagt cacgatggat ttccaaatta gtgtaggagg
     1981 ggggagggga gggagggaga taatgtccag gctgccataa gtggggaata aggaaaataa
     2041 aacatgaaac acgggtcggg caatgtcatg cggtattcgg ctttgctttc cggcgcgcct
     2101 aggccggccg acatgtacag agctcgagga gcgccggagt ataaatagag gcgcttcgtc
     2161 tacggagcga caattcaatt caaacaagca aagtgaacac gtcgctaagc gaaagctaag
     2221 caaataaaca agcgcagctg aacaagctaa acaatctgca gccaagctga tcctctaggg
     2281 tacgcgctag agtcgagagg cctgtttaaa cgatccaccg gtcgccacca tggtgagcaa
     2341 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     2401 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     2461 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     2521 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     2581 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2641 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2701 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2761 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     2821 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     2881 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     2941 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3001 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagagca ggcacagaag
     3061 gcatcgccag cgctctagga gccgcaatcg cagccgaagt cgcagcagtg aacgaaaacg
     3121 ccgtcaacgg agccgaagtc gcagcagtga acgaagacgc tacttgtaca agtaaagcgg
     3181 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     3241 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     3301 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3361 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttac
     3421 tagtggccac gtggaggccg gccgaattcg aatggccatg ggacgtcgac ctgtggtaat
     3481 tataaccctg gcgcctaagg atcgatactt ggggataccc tgaaccctct tcttccaaca
     3541 ccaaatgcaa atatatatac agactctata tatatgcaga ctctagctct tattatttac
     3601 ccaatttgct caaaaaacaa gatttcgata tcgattttta tcgattgctt ggaaacgggg
     3661 caagttatcg aatatcggaa acaaactcga tctgcgtaga cactagtcaa gttcttcaag
     3721 tctctagctc ttctgatatc cctgccttca tacatatgga cggacggacg gacggacatg
     3781 gctagatcga ctcggctatt gatgctgatc aagaatatat atactttatg gggtcggagg
     3841 tgcttcattc tgcctgttac atacatttgc attttgcaca aacccttctt acccactttc
     3901 aatgggttca gagtataaaa tgaaattgtt tttgtgctca tcgaattcga aattgtcatt
     3961 caagcagaaa attgaaatca actgtgaaat ttccaataaa tattaagcat atacaattat
     4021 gcatttgaag ctattccaaa ttgaagctag aaattgagca tatctgcggc acgccgaaga
     4081 tgaaataccc ttcctttgca gataggcgca cattgtcacg cctcaattct catttctcga
     4141 ccgattcttt cctttgatct atattagata gtcgtacaat atgagatgaa ctagtcaatg
     4201 ctacagttag tccagatatc ttgatgaaaa caaagttttt cgtacaagaa cctacttagc
     4261 gacctatcgt tcctatgcca gctatatgat atagaaaacc agaagagatt acctacgacg
     4321 aattgcaaga cgttagctaa aatgctgaga gactagttct cagagaaaca aacagatgga
     4381 tcagatatat atatggagat gttcccttct aggtgttaca cattccatga caatctcata
     4441 ataccccgca caagcatcac tagtgaattc gcggccgcct gcaggtcgac catatgggag
     4501 agctcccaac gcgttggatg catagcttga gtattctata gtgtcaccta aatagcttgg
     4561 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4621 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4681 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4741 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4801 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4861 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4921 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4981 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5041 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5101 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5161 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5221 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5281 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5341 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5401 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5461 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5521 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5581 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5641 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5701 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5761 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5821 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5881 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5941 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6001 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6061 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6121 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6181 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6241 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6301 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6361 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6421 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6481 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6541 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6601 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6661 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6721 ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg
     6781 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     6841 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     6901 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     6961 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     7021 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     7081 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     7141 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     7201 ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg
     7261 ggaagggcga tcggtgcggg cctcttcgct
//
