LOCUS       sequence-240659-        6851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas mediated knock-in via non-homologous end-joining in 
            Tribolium; bhsp drives EGFP expression; Dm-ebony for linearization; 
            (Transformation plasmid; Black eye marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6851)
  AUTHORS   He B, Buescher M, Farnworth MS, Strobl F, Stelzer EH, Koniszewski 
            ND, Muehlen D, Bucher G
  TITLE     An ancestral apical brain region contributes to the central complex 
            under the control of foxQ2 in the beetle Tribolium.
  JOURNAL   Elife. 2019 Oct 18;8. pii: 49065. doi: 10.7554/eLife.49065.
  PUBMED    31625505
REFERENCE   2  (bases 1 to 6851)
  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: "Elife."; 
            date: "2019-10-18"; volume: "8. pii: 49065. doi"; pages: " 
            10.7554/eLife.49065"
COMMENT     Sequence Label: pJET1.2[eb-Tc'bhsp-EGFP-2A-Cre;3xP3-gTc'v-SV40]
FEATURES             Location/Qualifiers
     source          1..6851
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(67..858,859..927))
                     /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     690..709
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(928..1032)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1100..1118
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     1424..1443
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1424..1442
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3150..3169)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3179..3260
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3204..3223
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3365..4396)
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             complement(4397..4453)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(join(4463..5173,5174..5176,5177..5179))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(4463..5173,5174..5176,5177..5179))
                     /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(4505..4526)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4854..4873
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(5767..5789)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5778..5794
                     /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(5802..5808,5809..5826,5827..5832))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    5847..5868
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5988..6005)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6159..6747)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6239..6258)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
       61 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
      121 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
      181 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
      241 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
      301 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
      361 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
      421 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
      481 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
      541 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
      601 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
      661 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
      721 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
      781 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
      841 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
      901 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
      961 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     1021 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     1081 catgacatta acctataaaa ataggcgtat cacgaggccg cccctgcagc cgaattatat
     1141 tatttttgcc aaataatttt taacaaaagc tctgaagtct tcttcattta aattcttaga
     1201 tgatacttca tctggaaaat tgtcccaatt agtagcatca cgctgtgagt aagttctaaa
     1261 ccattttttt attgttgtat tatctctaat cttactactc gatgagtttt cggtattatc
     1321 tctattttta acttggagca ggttccattc attgtttttt tcatcatagt gaataaaatc
     1381 aactgcttta acacttgtgc ctgaacacca tatccatccg gcgtaatacg actcactata
     1441 gggagagcgg ccgccagatc ttccggatgg ctcgagtttt tcagcaagat tctagacatt
     1501 attcattaga gactaattca attagagcta attcaattag gatccaagct tatcgatttc
     1561 gaaccctcga ccgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt
     1621 caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg
     1681 aacaagctaa acaatcgggg taccgctaga gtcgacggta ccgcggtcgt aatgagttgc
     1741 ccactgagac cctcgtaagt atgatttaat ttaattattc catttacaat taatcgtaaa
     1801 ttaagtgaag cccaagaagg cgaccaactg agcgaagaat gtgggatgct ttacggtgaa
     1861 tacttgatgt tggacaagat tttagaagcc caaagactac tcagcgagca aaataaccaa
     1921 cccgtgcacg acgagcactt atttattgtg acccatcaag gtaaaaaacc cacttaaccg
     1981 taattaacta taacaagtaa ttccagcgta tgagttatgg ttcaaacaaa taatctacga
     2041 gctggactcg atccgcaaca ttttcagcga cgttttagaa gagtcgcaaa ctttggaaat
     2101 cctcaaacgc ctcaatcgtg ttgttttgat cctcaaggtt cacaatttcg caacaaattg
     2161 cggtcacgag ataaaactgt cgcaattgta ggttttggtg gaccaagtaa tgattctgga
     2221 aacgatgaca ccgctcgatt tcatggactt ccggtgttat ctgcggcccg cttccgggtt
     2281 ccagagcctg cagttcaggc tcctggaaaa taagctcggg gtgcgccagg aaaaccgcgt
     2341 caaatacaac cagaattact cgaaagtgtt cggaaacgac gaaaaagcgc tcgagcaaat
     2401 cgccaagtcg gagaaagagc cgtctttgac cgatttggtc cagcggtggc tggagcgaac
     2461 gccggggctg gagctcgagg ggtttaattt ctgggggaag taccaaaaag cggtcgagaa
     2521 gcttttaacc gagcaaaaag aactggcgga gaaagaggag gcggagactt taaagcgtta
     2581 caaattgaac gatttggaga agagacgcga ggtttacgag tcgattttta aagttgaggt
     2641 ccatgaggct ctaatgtcgc gcggcgagag gcgcttctca cacaaagccc tgcaaggggc
     2701 tatcatgatc accttttaca gggacgaacc caggttcagt cagccccacc aaattctaac
     2761 acttttgatg gacatagact cgctgattac gaaatggcgg tgttagtaaa tccgtacgtc
     2821 aattgttttt gtttaaattt tataatttca gataatcatg ttttgatggt gcaaagaatg
     2881 attggttctt cgcagttagg aacagggggc tcttcggggt atcagtattt aaggtcaacg
     2941 ttaaggtgaa attgatttca acacggtttt tgcaatgttt gattttgttt ttcagcgatc
     3001 ggtataaagt tttcgtggac cttttcaatt tatcgacgtt tttgatacca cggtcctaca
     3061 tccccccctt gtctacgtct atgagaagtc atttgtgcaa ttgggggtcg gcgaatagca
     3121 ccaataacgt gtcgaatgga aacaattaag atgagtttgg acaaaccaca actagaatgc
     3181 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     3241 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     3301 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt
     3361 atgactaatc gccatcttcc agcaggcgca ccattgcccc tgtttcacta tccaggttac
     3421 ggatatagtt catgacaata tttacattgg tccagccacc agcttgcatg atctccggta
     3481 ttgaaactcc agcgcgggcc atatctcgcg cggctccgac acgggcactg tgtccagacc
     3541 aggccaggta tctctgacca gagtcatcct tagcgccgta aatcaatcga tgagttgctt
     3601 caaaaatccc ttccagggcg cgagttgata gctggctggt ggcagatggc gcggcaacac
     3661 cattttttct gacccggcaa aacaggtagt tattcggatc atcagctaca ccagagacgg
     3721 aaatccatcg ctcgaccagt ttagttaccc ccaggctaag tgccttctct acacctgcgg
     3781 tgctaaccag cgttttcgtt ctgccaatat ggattaacat tctcccaccg tcagtacgtg
     3841 agatatcttt aaccctgatc ctggcaattt cggctatacg taacagggtg ttataagcaa
     3901 tccccagaaa tgccagatta cgtatatcct ggcagcgatc gctattttcc atgagtgaac
     3961 gaacctggtc gaaatcagtg cgttcgaacg ctagagcctg ttttgcacgt tcaccggcat
     4021 caacgttttc ttttcggatc cgccgcataa ccagtgaaac agcattgctg tcacttggtc
     4081 gtggcagccc ggaccgacga tgaagcatgt ttagctggcc caaatgttgc tggatagttt
     4141 ttactgccag accgcgcgcc tgaagatata gaagataatc gcgaacatct tcaggttctg
     4201 cgggaaacca tttccggtta ttcaacttgc accatgccgc ccacgaccgg caaacggaca
     4261 gaagcatttt ccaggtatgc tcagaaaacg cctggcgatc cctgaacatg tccatcaggt
     4321 tcttgcgaac ctcatcactc gttgcatcga ccggtaatgc aggcaaattt tggtgtacgg
     4381 tcagtaaatt ggacatagga ccggggtttt cttccacgtc tcctgcttgc tttaacagag
     4441 agaagttcgt ggctccggat cccttgtaca gctcgtccat gccgagagtg atcccggcgg
     4501 cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg
     4561 cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg
     4621 tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct
     4681 tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt
     4741 tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct
     4801 tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt
     4861 agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg
     4921 acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt
     4981 aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga
     5041 acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact
     5101 tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct
     5161 cctcgccctt gctcaccatg tttgactttg aattcactag taaataattc actcaacttt
     5221 gttaaagtcg ctttgaaaat ttgctttgag tcgcttgctt agctttgttg ctttcgcttc
     5281 gcttgattca aattcactga caacgcgccg cgagaccgcg cttatatatg aaacgccagg
     5341 aggcgggctg cccggttcgg gcccgacatc tttctagaag atctcctaca atattctcag
     5401 ctgccatgga aaatcgatgt tcttctttta ttctctcaag attttcaggc tgtatattaa
     5461 aacttatatt aagaactatg ctaaccacct catcaggaac cgttgtaggt ggcgtgggtt
     5521 ttcttggcaa tcgactctca tgaaaactac gagctaaata ttcaatatgt tcctcttgac
     5581 caactttatt ctgcattttt tttgaacgag gtttagagca agcttcagga aactgagaca
     5641 ggaattttat taaaaattta aattttgaag aaagttcagg gttaatagca tccatttttt
     5701 gctttgcaag ttcctcagca ttcttaacaa aagacgtctc ttttgacatg tttaaagttt
     5761 aaacctcctg tgtgaaattg ttatccgctc acaattccac acattatacg agccggaagc
     5821 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5881 tcactgccaa ttgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5941 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     6001 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6061 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6121 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6181 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6241 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6301 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6361 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6421 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6481 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6541 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6601 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6661 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6721 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6781 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6841 ttcacctaga t
//
