LOCUS       sequence-419843-        7769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rhno1 S162A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7769)
  AUTHORS   Brambati A, Sacco O, Porcella S, Heyza J, Kareh M, Schmidt JC, Sfeir
            A
  TITLE     RHINO directs MMEJ to repair DNA breaks in mitosis.
  JOURNAL   Science. 2023 Aug 11;381(6658):653-660. doi: 
            10.1126/science.adh3694. Epub 2023 Jul 13.
  PUBMED    37440612
REFERENCE   2  (bases 1 to 7769)
  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"; doi: 
            "10.1126/science.adh3694"; journalName: "Science"; date: 
            "2023-08-11- 11"; volume: "381"; issue: "6658"; pages: "653-660"
COMMENT     Sequence Label: pLenti-h.Rhno1 S162A (D)-C-Myc-DDK-P2A-Puro
FEATURES             Location/Qualifiers
     source          1..7769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             834..1014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1061..1186
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1683..1916
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2101..2145
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2443..2560
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2643..3022
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3023..3226
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3252..3271
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3252..3270
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4061..4090
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4109..4132
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4151..4207
                     /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             4208..4807
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4208..4227)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4704..4724
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(4864..4897)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4953..5541
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5006..5026)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5424..5435)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5544..5560)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5545..5561)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6070..6250
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6312..6900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6392..6411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6977..7636)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7551..7570
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7637..7741)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     1021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     1081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     1201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     1261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     1321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     1381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     1441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     1501 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat
     1561 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1621 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1681 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1741 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1801 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1861 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1921 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1981 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     2041 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     2101 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     2161 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     2221 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2281 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2341 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2401 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2461 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2521 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2581 cagagatcca gtttggttag taccgggccc gctctagaca tgtccaatat gaccgccatg
     2641 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     2701 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2761 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2821 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2881 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc
     2941 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt
     3001 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata
     3061 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     3121 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca
     3181 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     3241 tcagaatttt gtaatacgac tcactatagg gcggccggga attcgtcgac tggatccggt
     3301 accgaggaga tctgccgccg cgatcgccat gcctcccaga aaaaaacgcc gccagccttc
     3361 ccagaaagcc ccgctgctgt tccaccaaca accactggag ggccccaaac acagctgtgc
     3421 atctacacag cttcccatca ctcacactcg acaggtgccc agcaagccca ttgaccacag
     3481 caccatcact tcctgggtat cacctgattt tgatacagca gcaggaagct tgttcccagc
     3541 ctaccagaaa caccaaaacc gggcgagaca ctcaagtcga aaacctacca cctccaagtt
     3601 tccacatcta acttttgaga gtccgcaatc ttccagttca gagacattgg ggatcccctt
     3661 aatccgagag tgccccagtg aatcagaaaa ggatgtttcc agaagaccct tagttccagt
     3721 gctcagtccc caaagctgtg ggaacatgtc agtgcaggca cttcagagct taccttatgt
     3781 gttcattcca cctgatatcc agaccccaga ggcctcatct gtgaaggaag aactcattcc
     3841 ccaagatcag aaggaaaaca gccttctaag ctgcactctt cacactggca ctcctaatag
     3901 cccagagcct ggacctgttc tggttaaaga cacccccgag gacaagtatg gaataaaggt
     3961 cacatggagg agacgacagc acctgcttgc ttacctcagg gagagaggga agctgagcag
     4021 aagccaattc cttgtgaaaa gcacgcgtac gcggccgctc gagcagaaac tcatctcaga
     4081 agaggatctg gcagcaaatg atatcctgga ttacaaggat gacgacgata aggtttgggt
     4141 aggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc
     4201 tggacctatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     4261 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     4321 tccggatcgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     4381 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     4441 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     4501 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     4561 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     4621 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     4681 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     4741 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     4801 tgcctgatgt acacccaaac ggccggccgc ggtctgtaca agtaggattc gtcgagggac
     4861 ctaataactt cgtatagcat acattatacg aagttataca tgtttaaggg ttccggttcc
     4921 actaggtaca attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg
     4981 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     5041 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     5101 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     5161 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     5221 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     5281 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     5341 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     5401 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     5461 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     5521 ccctttgggc cgcctccccg catcgatacc gtcgacctcg atcgagacct agaaaaacat
     5581 ggagcaatca caagtagcaa tacagcagct accaatgctg attgtgcctg gctagaagca
     5641 caagaggagg aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact
     5701 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     5761 attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac
     5821 ttccctgatt ggcagaacta cacaccaggg ccagggatca gatatccact gacctttgga
     5881 tggtgctaca agctagtacc agttgagcaa gagaaggtag aagaagccaa tgaaggagag
     5941 aacacccgct tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagta
     6001 ttagagtgga ggtttgacag ccgcctagca tttcatcaca tggcccgaga gctgcatccg
     6061 gactgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     6121 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     6181 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     6241 atctctagca gcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     6301 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     6361 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6421 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6481 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6541 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6601 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6661 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6721 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6781 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6841 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6901 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6961 gggattttgg tcatgattac gccccgccct gccactcatc gcagtactgt tgtaattcat
     7021 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
     7081 gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga
     7141 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
     7201 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
     7261 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc
     7321 agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
     7381 cccatatcac cagctcaccg tctttcattg ccatacggaa ctccggatga gcattcatca
     7441 ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct
     7501 ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact
     7561 gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag
     7621 tgattttttt ctccatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7681 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7741 gcacatttcc ccgaaaagtg ccacctgac
//
