LOCUS       sequence-366119-        6967 bp    DNA     circular SYN 15-DEC-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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6967)
  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 6967)
  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: "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..6967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     primer_bind     609..628
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        609..627
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(650..837,838..849)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(903..922)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             914..1723
                     /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(981..1000)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1866..2454
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2355..2374
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2608..2625
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(2655..2666,2667..2829)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     CDS             2955..3011
                     /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(5779..5781,5782..5784,5785..6498)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5824..5845)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6085..6104)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6432..6453
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6736..6753)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6742..6967
                     /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 gcaaggaagc gccgggccct cgtatggagt
      541 caaattatgc ggcagggagt tcatccgaaa ccgctgtggc ccgaacgcct cccgctagcg
      601 atgggatcta atacgactca ctatagggag accaagggaa aatagaatga agtgatctcc
      661 aaaaaataag tactttttga ctgtaaataa aattgtaagg agtaaaaagt actttttttt
      721 ctaaaaaaat gtaattaagt aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag
      781 taaaaatccc caaaaataat acttaagtac agtaatcaag taaaattact caagtacttt
      841 acacctctgg gatccagctg catcatgaac aataaaactg tctgcttaca taaacagtaa
      901 tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa
      961 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
     1021 gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
     1081 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
     1141 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac
     1201 cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
     1261 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     1321 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa
     1381 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     1441 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     1501 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     1561 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     1621 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
     1681 gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
     1741 gttgtaacac tggcaaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1801 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1861 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1921 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1981 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     2041 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2101 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2161 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2221 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacggttccc
     2281 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2341 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2401 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2461 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2521 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2581 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaacagatt
     2641 taaatggtac cgagcagagg tgtaaaaagt actcaaaaat tttactcaag tgaaagtaca
     2701 agtacttagg gaaaatttta ctcaattaaa agtaaaagta tctggctaga atcttacttg
     2761 agtaaaagta aaaaagtact ccattaaaat tgtacttgag tattaaggaa gtaaaagtaa
     2821 aagcaagaaa gaaaactaga gtggtctccc tttagtgagg gtcaattgat gggaggcgtt
     2881 cgggccacag cggtttctcg gctctcgtag tgtgtctgct gctggctgga gtaaaggcgc
     2941 tggaccttcc cggagccacg aacttctctc tgttaaagca agcaggagac gtggaagaaa
     3001 accccggtcc tatgccaccc aagcgcaaaa cgcttaacgc tgcggctgag gctaacgctc
     3061 atgccgacgg acacgccgac ggaaacgccg acggacacgt ggccaatacg gccgcgtcct
     3121 cgaataatgc gaggttcgct gatctcacta acatcgatac tccgggtctg ggacccacaa
     3181 ctacgaccct gctcgtggaa ccagcacgct caaagcgtca acgagtgtcc cgcgcatgcg
     3241 accagtgccg tgcagcccga gagaaatgcg acggaataca gcctgcgtgt ttcccgtgcg
     3301 tttcccaggg aaggtcctgc acttatcagg cttcgccgaa aaagagggga gttcaaaccg
     3361 gttatattcg tacgctggag ctcgccctcg cctggatgtt tgaaaatgtc gcgcgttccg
     3421 aagatgcctt gcataacctc ctcgtccgtg acgccggaca aggatcagct ctgctcgttg
     3481 gtaaagattc gccggctgcc gagcgactcc atgcccgttg ggctactagc cgtgtcaata
     3541 agagcattac ccgcctcctc cgtcagttgg agctccctcc taccgccacg gctacggcct
     3601 cgataatgcc gcacgtgatg gagcagcctc tcagtaccag cattaacccc gtcaacgacc
     3661 gcttcaacgg tattcccaac cccactccgt ataactccga tgcagctctc gatgctatca
     3721 ctcagaccaa cgattatgga agcgtaaata cacatggtat cctctctact tacccgccac
     3781 cggctacgca ccttaatgaa gcttccgtcg ctctcgctcc cggtggcgcc cccccccgac
     3841 cgcctcctcc gtatgttgac agcacgacca atcacccgcc gtaccactcg aatctggttc
     3901 caatggcgaa ctttggttac tcgaccgttg attacgatgc catggttgac gatttggcta
     3961 gcattgaata cacggacgct gtggatgtcg acccacagtt tatgaccaat ctgggattcg
     4021 ttcctggatg taacttctcc gacattaata catacgaaca gtgaaactag taattaagtc
     4081 tcagccaccg ttaactgaac atgtcaaaac ctgtggagac tgttgagatt tgatgttctg
     4141 aaaagataaa gcctataaat aaaatgttgc ccaaatttcc tgcctgatgt ttttctttgt
     4201 ctttgctaca tggctttgct gctcggatcg gctcactctg tgtatgccac gttcactttg
     4261 tactctcctt ctcacggtag gtttattatt tttagatgtg cagttagttt ctgtgaaata
     4321 acacaccaca cactgatatt gtctgtgcat tgacttggtg agtgcacatt gtttttgatc
     4381 ttgacatatt tatatttgat tgatcaggtg aactgtgtga atctaaagtg ctccatacag
     4441 atgttctgca ttgaaaatat tctcatttta ttagtggaag tgagtgtatg ccacatccaa
     4501 tcaatttcag caaacacccc agtatgattt aatgcaaaaa aatgaaggta tcaaacacgc
     4561 attactactt tgcagttaaa tatttaacat ttattccaac acgaaaaaaa gcagtaaata
     4621 acactttgac aaacacgtca ggacatctta tttttgtcac cctcacaggc aatttagtat
     4681 aatatattat atatatatat atatcatata ataatattca gtataatata tatatatata
     4741 tcatattata atattcagta taatataaaa cacaaacaca tatatgtata atataatata
     4801 acatttttat ttattgagat gcctctatgg accgtgttat aagaagtaaa gatcaggaga
     4861 agtaaacatg aagtgtaatt atgaatactg atgttaaatt aagctatgat gagttttcac
     4921 tgttaattta ccatctcaat taaatgttga tgcctccatg accaagttaa gcagatgaga
     4981 ctgaggacaa ctgtagaaga caagatgttc actttgctga atatagctgg cttgacagtt
     5041 atctatgact ctataaatat atatatattt ttttttataa aatgatttat ttataactat
     5101 atatccattt ctcagacagg tgcttcatat ccctcactcc cgtagctgtc catgctggat
     5161 ctgtccccgt tgtttttaaa aagctaaata agttattaac atgactgcat ccagcgagcc
     5221 aaacctgtct ggtgtacagc taccagagaa gcttgagatc ctagtcaccg gcggcgttcc
     5281 tagttcttta aactcaacca ctccaggcat agctagagcc actgatgcat ctatagagct
     5341 gaagtaaaga tagggttgat gcaaatcgat ttagaatttt caatgtagaa aaaacaaaat
     5401 aaaaagtcta ttcacttgat ggatagatta gtcaaacaaa catagagtag gcacttccct
     5461 tgactctgta aacttttgga tttaacagat tgtgcactcc ttttagttaa acacattagt
     5521 ccctgaaaac taagacccta accataatcc cattgtagac ccacctactc gggtgaatat
     5581 aaagctcgca cagatcatct cacagacatc agaccttggc aaaggagcat cctgaacatc
     5641 tctacacact gaggcaaccc tagggccgcc accatgctgt gttgtatcag aagaacaaag
     5701 cctgtggaga agaatgagga ggctgaccag gaactgcaga gcacggtacc tagagctaga
     5761 gaccctcctg tggctacaat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     5821 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     5881 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     5941 cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg cttcgcccgc
     6001 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     6061 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     6121 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     6181 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     6241 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     6301 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     6361 ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga ccccaacgag
     6421 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     6481 gacgagctgt ataagtgaga attctgcagg aattgggtgg catccctgtg acccctcccc
     6541 agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa
     6601 attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg
     6661 ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcacgag ggggggcccg
     6721 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     6781 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     6841 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     6901 aggacagcaa gggggaggat tgggaagaca atagcaaggc atgctgggga tgcggtgggc
     6961 tctatgg
//
