LOCUS       sequence-358680-        7395 bp    DNA     linear   UNA 15-APR-2025
DEFINITION  Modified version of GeneWeld method from Wierson et al., 2020 
            (https://doi.org/10.7554/eLife.53968). Contains homology arm inserts
            for integration of QF2 into rln3a gene by CRISPR/Cas9 integration..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7395)
  AUTHORS   Spikol ED, Cheng J, Macurak M, Subedi A, Halpern ME
  TITLE     Genetically defined nucleus incertus neurons differ in connectivity 
            and function.
  JOURNAL   Elife. 2024 May 31;12:RP89516. doi: 10.7554/eLife.89516.
  PUBMED    38819436
REFERENCE   2  (bases 1 to 7395)
  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: "Elife."; 
            date: "2024-05-31"; volume: "12:RP89516. doi"; pages: " 
            10.7554/eLife.89516"
COMMENT     Sequence Label: pPRISM-QF2-he1.1:YFP-rln3a-HA
FEATURES             Location/Qualifiers
     source          1..7395
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     polyA_signal    70..151
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    275..356
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        673..691
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(967..986)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             978..1787
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1045..1064)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1930..2518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2419..2438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2672..2689
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2980..3001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3016..3021,3022..3039,3040..3046)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3054..3070
                     /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)."
     primer_bind     3059..3081
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3078..3094
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3078..3094
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3090..3467
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSYATQAQNA"
     promoter        3115..3133
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(3181..3199)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3209..3226)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3209..3225)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3218..3240)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3385..3441
                     /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             join(6207..6209,6210..6212,6213..6926)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6252..6273)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6513..6532)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6860..6881
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7164..7181)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7170..7395
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cttctgaggc ggaaagaacc aaagggcgaa ttaattcatt gatgagtttg gacaaaccac
       61 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
      121 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
      181 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
      241 tcattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
      301 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
      361 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
      421 gcaagtaaaa cctctacaaa tgtggtcatt cagatcaatt aaccctcacc cgggctagcg
      481 atgcatggga gctctgacgt acgttgtcta gcaaggaagt gaagagctat ccggatatat
      541 atgtatgtta tcataactgt actggctatg tcaggtggta atgactgtaa cgttacgtta
      601 ctcgttgtag gcacgacatt gaatatgctc ttctccgctg tggcccgaac gcctcccgct
      661 agcgatggga tctaatacga ctcactatag ggagaccaag ggaaaataga atgaagtgat
      721 ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt
      781 ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt
      841 aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta
      901 ctttacacct ctgggatcca gctgcatcat gaacaataaa actgtctgct tacataaaca
      961 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt
     1021 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     1081 gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     1141 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     1201 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     1261 tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag
     1321 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     1381 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     1441 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     1501 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     1561 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     1621 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     1681 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     1741 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta
     1801 attggttgta acactggcaa aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1861 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1921 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1981 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2041 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     2101 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2161 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2221 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2281 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacggt
     2341 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2401 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2461 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2521 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2581 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2641 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaca
     2701 gatttaaatg gtaccgagca gaggtgtaaa aagtactcaa aaattttact caagtgaaag
     2761 tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc tagaatctta
     2821 cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattaa ggaagtaaaa
     2881 gtaaaagcaa gaaagaaaac tagagtggtc tccctttagt gagggtcaat tgatgggagg
     2941 cgttcgggcc acagcggaca cgcaggtgcg caacgcaatt aatgtgagtt agctcactca
     3001 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     3061 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     3121 ccctcactaa agggaacaaa agctggagga cctcgagggg gggcccggta cccaattcgc
     3181 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     3241 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     3301 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     3361 aatggacctg ccacacttcc cggagccacg aacttctctc tgttaaagca agcaggagac
     3421 gtggaagaaa accccggtcc tatgccaccc aagcgcaaaa cgcttaacgc tgcggctgag
     3481 gctaacgctc atgccgacgg acacgccgac ggaaacgccg acggacacgt ggccaatacg
     3541 gccgcgtcct cgaataatgc gaggttcgct gatctcacta acatcgatac tccgggtctg
     3601 ggacccacaa ctacgaccct gctcgtggaa ccagcacgct caaagcgtca acgagtgtcc
     3661 cgcgcatgcg accagtgccg tgcagcccga gagaaatgcg acggaataca gcctgcgtgt
     3721 ttcccgtgcg tttcccaggg aaggtcctgc acttatcagg cttcgccgaa aaagagggga
     3781 gttcaaaccg gttatattcg tacgctggag ctcgccctcg cctggatgtt tgaaaatgtc
     3841 gcgcgttccg aagatgcctt gcataacctc ctcgtccgtg acgccggaca aggatcagct
     3901 ctgctcgttg gtaaagattc gccggctgcc gagcgactcc atgcccgttg ggctactagc
     3961 cgtgtcaata agagcattac ccgcctcctc cgtcagttgg agctccctcc taccgccacg
     4021 gctacggcct cgataatgcc gcacgtgatg gagcagcctc tcagtaccag cattaacccc
     4081 gtcaacgacc gcttcaacgg tattcccaac cccactccgt ataactccga tgcagctctc
     4141 gatgctatca ctcagaccaa cgattatgga agcgtaaata cacatggtat cctctctact
     4201 tacccgccac cggctacgca ccttaatgaa gcttccgtcg ctctcgctcc cggtggcgcc
     4261 cccccccgac cgcctcctcc gtatgttgac agcacgacca atcacccgcc gtaccactcg
     4321 aatctggttc caatggcgaa ctttggttac tcgaccgttg attacgatgc catggttgac
     4381 gatttggcta gcattgaata cacggacgct gtggatgtcg acccacagtt tatgaccaat
     4441 ctgggattcg ttcctggatg taacttctcc gacattaata catacgaaca gtgaaactag
     4501 taattaagtc tcagccaccg ttaactgaac atgtcaaaac ctgtggagac tgttgagatt
     4561 tgatgttctg aaaagataaa gcctataaat aaaatgttgc ccaaatttcc tgcctgatgt
     4621 ttttctttgt ctttgctaca tggctttgct gctcggatcg gctcactctg tgtatgccac
     4681 gttcactttg tactctcctt ctcacggtag gtttattatt tttagatgtg cagttagttt
     4741 ctgtgaaata acacaccaca cactgatatt gtctgtgcat tgacttggtg agtgcacatt
     4801 gtttttgatc ttgacatatt tatatttgat tgatcaggtg aactgtgtga atctaaagtg
     4861 ctccatacag atgttctgca ttgaaaatat tctcatttta ttagtggaag tgagtgtatg
     4921 ccacatccaa tcaatttcag caaacacccc agtatgattt aatgcaaaaa aatgaaggta
     4981 tcaaacacgc attactactt tgcagttaaa tatttaacat ttattccaac acgaaaaaaa
     5041 gcagtaaata acactttgac aaacacgtca ggacatctta tttttgtcac cctcacaggc
     5101 aatttagtat aatatattat atatatatat atatcatata ataatattca gtataatata
     5161 tatatatata tcatattata atattcagta taatataaaa cacaaacaca tatatgtata
     5221 atataatata acatttttat ttattgagat gcctctatgg accgtgttat aagaagtaaa
     5281 gatcaggaga agtaaacatg aagtgtaatt atgaatactg atgttaaatt aagctatgat
     5341 gagttttcac tgttaattta ccatctcaat taaatgttga tgcctccatg accaagttaa
     5401 gcagatgaga ctgagacaac tgtagaagac aagatgttca ctttgctgaa tatagctggc
     5461 ttgacagtta tctatgactc tataaatata tatatatttt tttttttata aaatgattta
     5521 tttataacta tatatccatt tctcagacag gtgcttcata tccctcactc ccgtagctgt
     5581 ccatgctgga tctgtccccg ttgtttttaa aaagctaaat aagttattaa catgactgca
     5641 tccagcgagc caaacctgtc tggtgtacag ctaccagaga agcttgagat cctagtcacc
     5701 ggcgttccta gttctttaaa ctcaaccact ccaggcatag ctagagccac tgatgcatct
     5761 atagagctga agtaaagata gggttgatgc aaatcgattt agaattttca atgtagaaaa
     5821 aacaaaataa aaagtctatt cacttgatgg atagattagt caaacaaaca tagagtaggc
     5881 acttcccttg actctgtaaa cttttggatt taacagattg tgcactcctt ttagttaaac
     5941 acattagtcc ctgaaaacta agaccctaac cataatccca ttgtagaccc acctactcgg
     6001 gtgaatataa agctcgcaca gatcatctca cagacatcag accttggcaa aggagcatcc
     6061 tgaacatctc tacacactga ggcaacccta gggccgccac catgctgtgt tgtatcagaa
     6121 gaacaaagcc tgtggagaag aatgaggagg ctgaccagga actgcagagc acggtaccta
     6181 gagctagaga ccctcctgtg gctacaatgg tgagcaaggg cgaggagctg ttcaccgggg
     6241 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     6301 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     6361 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgcagtgct
     6421 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     6481 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     6541 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     6601 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     6661 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     6721 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     6781 gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc
     6841 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     6901 tcggcatgga cgagctgtat aagtgagaat tctgcaggaa ttgggtggca tccctgtgac
     6961 ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc
     7021 ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg
     7081 tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag ggcacgaggg
     7141 ggggcccgaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     7201 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     7261 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     7321 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaaggcat gctggggatg
     7381 cggtgggctc tatgg
//
