LOCUS       sequence-240664-        6876 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CRISPR/Cas mediated knock-in via non-homologous end-joining in 
            Tribolium; bhsp drives EGFP expression; Dm-ebony and Dm-yellow for 
            linearization; (Transformation plasmid; Black eye marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6876)
  TITLE     CRISPR plasimids for knock-in via non-homologous end-joining
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6876)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pJET1.2[eb-Tc'bhsp-EGFP-2A-Cre; ye-3xP3-gTc'v-...
FEATURES             Location/Qualifiers
     source          1..6876
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(89..880,881..949))
                     /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     712..731
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(950..1054)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1122..1140
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     1446..1465
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1446..1464
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1701..1703,1704..1706,1707..2417)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1701..1703,1704..1706,1707..2417)
                     /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(2007..2026)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2354..2375
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2427..2483
                     /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             2484..3515
                     /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"
     polyA_signal    3620..3701
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3657..3676)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3711..3730
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5814..5836)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5825..5841
                     /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(5849..5855,5856..5873,5874..5879))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    5894..5915
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6035..6052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6206..6794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6286..6305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
       61 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
      121 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
      181 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
      241 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
      301 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
      361 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
      421 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
      481 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
      541 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
      601 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
      661 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
      721 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
      781 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
      841 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
      901 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
      961 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     1021 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     1081 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     1141 cgcccctgca gccgaattat attatttttg ccaaataatt tttaacaaaa gctctgaagt
     1201 cttcttcatt taaattctta gatgatactt catctggaaa attgtcccaa ttagtagcat
     1261 cacgctgtga gtaagttcta aaccattttt ttattgttgt attatctcta atcttactac
     1321 tcgatgagtt ttcggtatta tctctatttt taacttggag caggttccat tcattgtttt
     1381 tttcatcata gtgaataaaa tcaactgctt taacacttgt gcctgaacac catatccatc
     1441 cggcgtaata cgactcacta tagggagagc ggccgccaga tcttccggat ggctcgagtt
     1501 tttcagcaag atgtcgggcc cgaaccgggc agcccgcctc ctggcgtttc atatataagc
     1561 gcggtctcgc ggcgcgttgt cagtgaattt gaatcaagcg aagcgaaagc aacaaagcta
     1621 agcaagcgac tcaaagcaaa ttttcaaagc gactttaaca aagttgagtg aattatttac
     1681 tagtgaattc aaagtcaaac atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1741 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1801 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1861 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1921 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1981 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     2041 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     2101 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     2161 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     2221 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     2281 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     2341 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2401 tggacgagct gtacaaggga tccggagcca cgaacttctc tctgttaaag caagcaggag
     2461 acgtggaaga aaaccccggt cctatgtcca atttactgac cgtacaccaa aatttgcctg
     2521 cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg
     2581 atcgccaggc gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg
     2641 cggcatggtg caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg
     2701 attatcttct atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg
     2761 gccagctaaa catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg
     2821 tttcactggt tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac
     2881 aggctctagc gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg
     2941 atcgctgcca ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac
     3001 gtatagccga aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa
     3061 tgttaatcca tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac
     3121 ttagcctggg ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg
     3181 atccgaataa ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca
     3241 ccagccagct atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt
     3301 acggcgctaa ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg
     3361 tcggagccgc gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg
     3421 gtggctggac caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag
     3481 gggcaatggt gcgcctgctg gaagatggcg attagtcata atcagccata ccacatttgt
     3541 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     3601 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     3661 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3721 caaactcatc ttaattgttt ccattcgaca cgttattggt gctattcgcc gacccccaat
     3781 tgcacaaatg acttctcata gacgtagaca agggggggat gtaggaccgt ggtatcaaaa
     3841 acgtcgataa attgaaaagg tccacgaaaa ctttataccg atcgctgaaa aacaaaatca
     3901 aacattgcaa aaaccgtgtt gaaatcaatt tcaccttaac gttgacctta aatactgata
     3961 ccccgaagag ccccctgttc ctaactgcga agaaccaatc attctttgca ccatcaaaac
     4021 atgattatct gaaattataa aatttaaaca aaaacaattg acgtacggat ttactaacac
     4081 cgccatttcg taatcagcga gtctatgtcc atcaaaagtg ttagaatttg gtggggctga
     4141 ctgaacctgg gttcgtccct gtaaaaggtg atcatgatag ccccttgcag ggctttgtgt
     4201 gagaagcgcc tctcgccgcg cgacattaga gcctcatgga cctcaacttt aaaaatcgac
     4261 tcgtaaacct cgcgtctctt ctccaaatcg ttcaatttgt aacgctttaa agtctccgcc
     4321 tcctctttct ccgccagttc tttttgctcg gttaaaagct tctcgaccgc tttttggtac
     4381 ttcccccaga aattaaaccc ctcgagctcc agccccggcg ttcgctccag ccaccgctgg
     4441 accaaatcgg tcaaagacgg ctctttctcc gacttggcga tttgctcgag cgctttttcg
     4501 tcgtttccga acactttcga gtaattctgg ttgtatttga cgcggttttc ctggcgcacc
     4561 ccgagcttat tttccaggag cctgaactgc aggctctgga acccggaagc gggccgcaga
     4621 taacaccgga agtccatgaa atcgagcggt gtcatcgttt ccagaatcat tacttggtcc
     4681 accaaaacct acaattgcga cagttttatc tcgtgaccgc aatttgttgc gaaattgtga
     4741 accttgagga tcaaaacaac acgattgagg cgtttgagga tttccaaagt ttgcgactct
     4801 tctaaaacgt cgctgaaaat gttgcggatc gagtccagct cgtagattat ttgtttgaac
     4861 cataactcat acgctggaat tacttgttat agttaattac ggttaagtgg gttttttacc
     4921 ttgatgggtc acaataaata agtgctcgtc gtgcacgggt tggttatttt gctcgctgag
     4981 tagtctttgg gcttctaaaa tcttgtccaa catcaagtat tcaccgtaaa gcatcccaca
     5041 ttcttcgctc agttggtcgc cttcttgggc ttcacttaat ttacgattaa ttgtaaatgg
     5101 aataattaaa ttaaatcata cttacgaggg tctcagtggg caactcatta cgaccgcggt
     5161 accgtcgact ctagcggtac cccgattgtt tagcttgttc agctgcgctt gtttatttgc
     5221 ttagctttcg cttagcgacg tgttcacttt gcttgtttga attgaattgt cgctccgtag
     5281 acgaagcgcc tctatttata ctccggcggt cgagggttcg aaatcgataa gcttggatcc
     5341 taattgaatt agctctaatt gaattagtct ctaatgaata atccagctgg ctccaactat
     5401 atcgctctag agacatcttt ctagaagatc tcctacaata ttctcagctg ccatggaaaa
     5461 tcgatgttct tcttttattc tctcaagatt ttcaggctgt atattaaaac ttatattaag
     5521 aactatgcta accacctcat caggaaccgt tgtaggtggc gtgggttttc ttggcaatcg
     5581 actctcatga aaactacgag ctaaatattc aatatgttcc tcttgaccaa ctttattctg
     5641 catttttttt gaacgaggtt tagagcaagc ttcaggaaac tgagacagga attttattaa
     5701 aaatttaaat tttgaagaaa gttcagggtt aatagcatcc attttttgct ttgcaagttc
     5761 ctcagcattc ttaacaaaag acgtctcttt tgacatgttt aaagtttaaa cctcctgtgt
     5821 gaaattgtta tccgctcaca attccacaca ttatacgagc cggaagcata aagtgtaaag
     5881 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgccaattg
     5941 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6001 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     6061 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     6121 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     6181 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6241 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6301 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6361 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6421 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6481 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6541 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6601 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6661 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6721 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6781 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6841 aaaactcacg ttaagggatt ttggtcatga gattat
//
