LOCUS       sequence-437008-        6415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector containing eGFP and mCherry reporter genes, for assembling an
            insulator-seq reporter library..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6415)
  AUTHORS   Tonelli A, Cousin P, Gambetta MC
  TITLE     Protocol for detecting genomic insulators in Drosophila using 
            insulator-seq, a massively parallel reporter assay.
  JOURNAL   STAR Protoc. 2024 Oct 24;5(4):103391. doi: 
            10.1016/j.xpro.2024.103391.
  PUBMED    39453817
REFERENCE   2  (bases 1 to 6415)
  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: "STAR 
            Protoc."; date: "2024-10-24"; volume: "5(4):103391. doi"; pages: " 
            10.1016/j.xpro.2024.103391"
COMMENT     Sequence Label: insulator_seq_reporter_vector
FEATURES             Location/Qualifiers
     source          1..6415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1013..1022
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          1028..1094
                     /label=Mhc intron
                     /note="modified Drosophila myosin heavy chain intron 16 
                     (Pfeiffer et al., 2010)"
     CDS             join(1109..1111,1112..1114,1115..1828)
                     /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(1109..1111,1112..1114,1115..1825)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1154..1175)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1415..1434)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1762..1783
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1844..1862
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     misc_feature    2560..2578
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             complement(2594..3304)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     3141..3159
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     intron          3319..3385
                     /label=Mhc intron
                     /note="modified Drosophila myosin heavy chain intron 16 
                     (Pfeiffer et al., 2010)"
     regulatory      3391..3400
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     terminator      4081..4128
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4216..4235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4426..4447
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4610..4714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4715..4783,4784..5575)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4933..4952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5746..6334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6235..6254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagtgttgt tggttggcac accacaaata tactgttgcc gagcacaatt gatcggctaa
       61 atggtatggc aagaaaaagt atgcaatata ataatctttt attgggtatg caacgaaaat
      121 ttgtttcgtc aaagtatgca atatttttta ttaaaagagg gtatgcaatg tattttatta
      181 aaaacgggta tgcaataaaa aattatttgg tttctctaaa aagtatgcag cacttatttt
      241 ttgataaggt atgcaacaaa attttacttt gccgaaaata tgcaatgttt ttgcgaataa
      301 attcaacgca cacttattac gtggccagga attcagatct gtggccagga attcattacg
      361 ccccaccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga
      421 agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt
      481 gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt
      541 ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa
      601 taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata
      661 tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag
      721 tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt
      781 ctttcattgc catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa
      841 aggccggata aaactccgga gtataaatag aggcgcttcg tcgacggagc gtcaattcaa
      901 ttcaaacaag caaagtgaac acatcgcgaa gcgtaagctg agcaaacaaa caagcgcagc
      961 tgaacaagct aaacaatctg cagaagcttg gcattccggt actgttggta aagccaccat
     1021 ggaaaaggta ggttcaacca ctgatgccta ggcacaccga aacgactaac cctaattctt
     1081 atcctttact tcaggccaaa gcttaagcat ggtgagcaag ggcgaggagc tgttcaccgg
     1141 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     1201 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     1261 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     1321 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1381 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     1441 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     1501 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     1561 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     1621 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     1681 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     1741 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1801 tctcggcatg gacgagctgt acaagtaagt ctcgtgggct cggagatgtg tataagagac
     1861 agtaagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     1921 tatttgtgaa atttgtgatg ctatttaggg gagaccatag cggccgactg aatccggtga
     1981 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     2041 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     2101 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     2161 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     2221 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     2281 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     2341 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     2401 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcggcc
     2461 gcactggtct ccttcgaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2521 ttctagttgt ggtttgtcca aactcatcaa tgtatcttac tgtctcttat acacatctcc
     2581 gagcccacga gacttacttg tacagctcgt ccatgccgcc ggtggagtgg cggccctcgg
     2641 cgcgttcgta ctgttccacg atggtgtagt cctcgttgtg ggaggtgatg tccaacttga
     2701 tgttgacgtt gtaggcgccg ggcagctgca cgggcttctt ggccttgtag gtggtcttga
     2761 cctcagcgtc gtagtggccg ccgtccttca gcttcagcct ctgcttgatc tcgcccttca
     2821 gggcgccgtc ctcggggtac atccgctcgg aggaggcctc ccagcccatg gttttcttct
     2881 gcattacggg gccgtcggag gggaagttgg tgccgcgcag cttcaccttg tagatgaact
     2941 cgccgtcctg cagggaggag tcctgggtca cggtcaccac gccgccgtcc tcgaagttca
     3001 tcacgcgctc ccacttgaag ccctcgggga aggacagctt caagtagtcg gggatgtcgg
     3061 cggggtgctt cacgtaggcc ttggagccgt acatgaactg aggggacagg atgtcccagg
     3121 cgaagggcag ggggccaccc ttggtcacct tcagcttggc ggtctgggtg ccctcgtagg
     3181 ggcggccctc gccctcgccc tcgatctcga actcgtggcc gttcacggag ccctccatgt
     3241 gcaccttgaa gcgcatgaac tccttgatga tggccatgtt atcctcctcg cccttgctca
     3301 ccatgcttaa gctttggcct gaagtaaagg ataagaatta gggttagtcg tttcggtgtg
     3361 cctaggcatc agtggttgaa cctacctttt ccatggtggc tttaccaaca gtaccggaat
     3421 gccaagcttc tgcagattgt ttagcttgtt cagctgcgct tgtttgtttg ctcagcttac
     3481 gcttcgcgat gtgttcactt tgcttgtttg aattgaattg acgctccgtc gacgaagcgc
     3541 ctctatttat actccggagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     3601 tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     3661 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     3721 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     3781 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     3841 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     3901 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     3961 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     4021 acaacagtac tgcgatgagt ggcagggtgg ggcgtaagga agagcggcgc attctagaaa
     4081 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttggg gaccggaaga
     4141 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4201 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4261 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4321 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     4381 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4441 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4501 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4561 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4621 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4681 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     4741 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4801 gaaagtaaaa gatgctgaag atcagttggg tgcacgggtg ggttacatcg aactggatct
     4861 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     4921 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4981 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5041 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5101 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5161 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5221 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     5281 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5341 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5401 tgctgataaa tctggagccg gtgagcgtgg gtcccgcggt atcattgcag cactggggcc
     5461 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5521 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5581 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5641 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5701 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5761 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5821 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5881 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5941 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6001 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6061 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6121 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6181 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6241 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6301 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6361 gttcctggcc ttttgctggc cttttgctca catgtcgtat gcttagctac gtgca
//
