LOCUS       sequence-306199-        4265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA for CRISPR/Cas9-mediated targeted integration into genomic 
            site T9 in CHO cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4265)
  AUTHORS   Sergeeva D, Lee GM, Nielsen LK, Grav LM
  TITLE     Multi-copy targeted integration for accelerated development of 
            high-producing CHO cells.
  JOURNAL   ACS Synth Biol. 2020 Aug 24. doi: 10.1021/acssynbio.0c00322.
  PUBMED    32835482
REFERENCE   2  (bases 1 to 4265)
  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: "ACS Synth 
            Biol. 2020 Aug 24. doi: 10.1021/acssynbio.0c00322."
COMMENT     Sequence Label: pRSFDuet1-sgRNA(siteT9)
FEATURES             Location/Qualifiers
     source          1..4265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        198..446
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     198..218
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     369..388
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        467..542
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(554..578)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        650..668
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    669..693
                     /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)."
     CDS             802..846
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(884..902)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      898..945
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1175..1990)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1175..1990)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1898..1917
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1991..2082)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2098..2847)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(2108..2125)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2348..2367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3043..4125)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4087..4106
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4126..4203)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4249..4265,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggtactg tacaaaaaag caggctttaa aggaaccaat tcagtcgact ggatccggta
      181 ccaaggtcgg gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat
      241 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
      301 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
      361 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
      421 atatcttgtg gaaaggacga aacaccgccc actacaggtt cgggcggttt tagagctaga
      481 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      541 gctttttttc tagacccagc tttcttgtac aaagttggca ttatgcggcc gcataatgct
      601 taagtcgaac agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc
      661 actatagggg aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa
      721 gaaggagata tacatatggc agatctcaat tggatatcgg ccggccacgc gatcgctgac
      781 gtcggtaccc tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg
      841 gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta
      901 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca
      961 tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag
     1021 acataagcgg ctatttaacg accctgccct gaaccgacga caagctgacg accgggtctc
     1081 cgcaagtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1141 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     1201 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     1261 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     1321 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     1381 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     1441 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     1501 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcggt
     1561 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     1621 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     1681 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     1741 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     1801 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     1861 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     1921 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     1981 tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     2041 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggcatg cagcgctctt
     2101 ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggtgtcag
     2161 ctcactcaaa agcggtaata cggttatcca cagaatcagg ggataaagcc ggaaagaaca
     2221 tgtgagcaaa aagcaaagca ccggaagaag ccaacgccgc aggcgttttt ccataggctc
     2281 cgcccccctg acgagcatca caaaaatcga cgctcaagcc agaggtggcg aaacccgaca
     2341 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2401 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2461 catagctcac gctgttggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2521 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2581 tccaacccgg taagacacga cttatcgcca ctggcagcag ccattggtaa ctgatttaga
     2641 ggactttgtc ttgaagttat gcacctgtta aggctaaact gaaagaacag attttggtga
     2701 gtgcggtcct ccaacccact taccttggtt caaagagttg gtagctcagc gaaccttgag
     2761 aaaaccaccg ttggtagcgg tggtttttct ttatttatga gatgatgaat caatcggtct
     2821 atcaagtcaa cgaacagcta ttccgttact ctagatttca gtgcaattta tctcttcaaa
     2881 tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc
     2941 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     3001 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     3061 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3121 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     3181 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     3241 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     3301 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
     3361 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     3421 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     3481 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     3541 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     3601 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     3661 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     3721 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     3781 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     3841 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     3901 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     3961 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     4021 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     4081 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     4141 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     4201 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaaatta atacgactca
     4261 ctata
//
