LOCUS       sequence-189642-        5931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a HR repair template for knock-in a YFP expression cassette 
            at the promoter of human NEAT1 gene. Best used with px330-NEAT1pr_v1
            vector, but also works with px330-NEAT1pr_v2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5931)
  TITLE     Unpublished Fox lab plasmids
REFERENCE   2  (bases 1 to 5931)
  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"
COMMENT     Sequence Label: pYFP-NEAT1pr_v1-RT
FEATURES             Location/Qualifiers
     source          1..5931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        703..1006
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1007..1210
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1161..1181
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1249..1258
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1255..1257,1258..1260,1261..1971)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1300..1321)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1561..1580)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1908..1929
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1972..2037
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2161..2282
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2198..2217)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2252..2271
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2847..3302)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2984..3005)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3196..3215
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3329..3433
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3435..3792
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3435..3626
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3458..3478)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3643..3778
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3827..4621
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3827..4621
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3881..3900)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4491..4510
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4809..4828)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4853..4900
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5229..5817
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5718..5737
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttatgga cgctatcagc ccgcagcagg gtttcctggc cagagaaacc gcctgttggg
       61 gtgcggagcg ttccacgtcc cctcgcagca ccccgctggc cttctgggcc gccccgcccc
      121 caacccgcgc ccgggcgctg acgtcatcgg ccgagcccga ctcggaacca ccgcccgaaa
      181 gtcacgcgcg cctcccggcg ggacgcgccc tctgcagaga tccctccgcc gccgcctgga
      241 attttccaga tgtcctgccg gcccttcttt cgggcctcgg ctgggcgtgg tgacctcaag
      301 cgaccccggc ggctcccgtc gcccactcaa gagggcgccc ccgcccgcca cgcccccagc
      361 ccctctcggg gaagcgcgtc ccccgcccga cctcaacaac atccgggaag aaaaggggtc
      421 ttcttcctca tggcattcgc ctcccaaatg tcaccttgag cgctgtccgc gatcccaaaa
      481 agcactgtta aagagaagcg gggatacact ggggtccttg cgtgggggcc gcctgggaga
      541 ccatgcaccg cccgggagtc tctccgggca gggtcgggga gggacttttt tgccggtggc
      601 cgtggaggaa tcgtcccgtt gagcaatgac cccggtgacg cggctgaggg catagtaatc
      661 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      721 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      781 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      841 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      901 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      961 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     1021 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     1081 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     1141 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     1201 aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc caccatggtg
     1261 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1321 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1381 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1441 accaccttcg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     1501 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1561 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1621 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1681 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1741 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1801 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1861 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1921 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggactc
     1981 agatctcgag ctcaagcttc gaattctgca gtcgacggta ccgcgggccc gggatccacc
     2041 ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     2101 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     2161 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2221 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2281 taattgtgga agtccagccg gagttagcga cagggaggga tgcgcgcctg ggtgtagttg
     2341 tgggggagga agtggctagc tcagggcttc aggggacaga cagggagaga tgactgagtt
     2401 agatgagact agggggcggg ctgggggtgc gagaaggaag cttggcaagg agactaggtc
     2461 tagggggacc acagtggggc aggctgcatg gaaaatatcc gcagggtccc ccaggcagaa
     2521 cagccacgct ccaggccagg ctgtccctac tgcctggtgg agggggaact tgacctctgg
     2581 gagggcgccg ctcttgcata gctgagcgag cccgggtgcg ctggtctgtg tggaaggagg
     2641 aaggcaggga gaggtagaag gggtggagga gtcaggagga ataggccgca gcagccctgg
     2701 aaatgatcag gaaggcaggc agtgggtgca gggctgcagg agggccggga gggctaatct
     2761 tcaacttgtc catgccagca gccccttttt ttccagacca agggctgtga acccgcctgg
     2821 ggatgaggcc tggtcttgtg gaacttaaat tgtaagcgtt aatattttgt taaaattcgc
     2881 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2941 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3001 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3061 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3121 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3181 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3241 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3301 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3361 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3421 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     3481 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3541 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3601 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3661 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3721 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3781 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3841 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3901 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3961 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     4021 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4081 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4141 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4201 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4261 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4321 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     4381 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4441 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4501 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4561 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4621 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4681 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4741 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg
     4801 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     4861 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     4921 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     4981 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     5041 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta
     5101 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5161 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5221 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5281 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5341 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5401 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5461 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5521 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5581 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5641 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5701 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5761 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5821 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5881 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
