LOCUS       sequence-320555-        5938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template for CAS9 complex genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5938)
  AUTHORS   Lo CA, Greben AW, Chen BE
  TITLE     Generating stable cell lines with quantifiable protein production 
            using CRISPR/Cas9-mediated knock-in.
  JOURNAL   Biotechniques. 2017 Apr 1;62(4):165-174. doi: 10.2144/000114534.
  PUBMED    28403807
REFERENCE   2  (bases 1 to 5938)
  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"; journalName: 
            "Biotechniques"; date: "2017-04-1- 1"; volume: "62"; issue: "4"; 
            pages: "165-174"
COMMENT     Sequence Label: 
            RPL13A_PQR_mScarlet_NOL_LoxP_PQR_zeo_PQR_MCS_RPL13A-3'UTR
FEATURES             Location/Qualifiers
     source          1..5938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             1158..1214
                     /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             1215..1910
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1348..1366)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1616..1635
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2024..2057)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             2067..2123
                     /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             2124..2495
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     protein_bind    complement(2496..2529)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             2541..2597
                     /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"
     primer_bind     complement(3767..3786)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3886..3908
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3946..3964)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4032..4136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4137..4205,4206..4997)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4355..4374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5168..5756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5657..5676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5910..5927
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccaga ggccaggccg ctctggaccg tctcaaggtg tttgacggca
      181 tcccaccgcc ctacgacaag gtgagctatg ccaaacccca caggcagcgg ccttacctgt
      241 ggcgtccatg atgttccgca actacctaca ttgtttgatc ctcatgaaag cagcactggc
      301 tgagacgcca gtccagccga cctccttccc tgtctgtccc tcccgggctc ttaagcccct
      361 ctctttctct aacagaaaaa gcggatggtg gttcctgctg ccctcaaggt cgtgcgtctg
      421 aagcctacaa gaaaggtgag tcccagctta cgctgcacca tctacttggg agatttcagg
      481 cctgctgagg gacctgggga cctggagcct ggcagatgat gtccttatct cacgatggtc
      541 tgcggatgtc cctgtgggaa tggcgacaat gccaatggct tagctgatgc caggaggctt
      601 gggtgggtgc ttttctaaca ggcctgcaga gaacagttgc attatgatat gcccagctgt
      661 cagtcacctc ccagctctca acagctccgg ctcttcaggg tgtgggggct tagatatcct
      721 tacaacttca tttgttcacc cccccccccc ccccccgcag tttgcctatc tggggcgcct
      781 ggctcacgag gttggctgga agtaccaggc agtgacagcc accctggagg agaagaggaa
      841 agagaaagcc aagatccact accggaagaa gaaacagctc atggtgaggc caggggctgg
      901 tgctgagggg ggcatctcac tcctggacag gcctggcagg tgccttgctc acagagtact
      961 cttaactggc aaaggaccag ccggggttgg ggtgggatgc agtccatgta atgagggcaa
     1021 tgcaacccct cctgaccacc accacctgca cttattcttg gcagaggcta cggaaacagg
     1081 ccgagaagaa cgtggagaag aaaattgaca aatacacaga ggtcctcaag acccacggac
     1141 tcctggtcgg aagcggagcg acgaatttta gtctactaaa acaagcgggt gatgtagaag
     1201 aaaaccctgg acctatggtg agcaagggcg aggcagtgat caaggagttc atgcggttca
     1261 aggtgcacat ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg
     1321 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     1381 ccttctcctg ggacatcctg tcccctcagt tcatgtacgg ctccagggcc ttcaccaagc
     1441 accccgccga catccccgac tactataagc agtccttccc cgagggcttc aagtgggagc
     1501 gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc tccctggagg
     1561 acggcaccct gatctacaag gtgaagctcc gcggcaccaa cttccctcct gacggccccg
     1621 taatgcagaa gaagacaatg ggctgggaag cgtccaccga gcggttgtac cccgaggacg
     1681 gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa ggacggcggc cgctacctgg
     1741 cggacttcaa gaccacctac aaggccaaga agcccgtgca gatgcccggc gcctacaacg
     1801 tcgaccgcaa gttggacatc acctcccaca acgaggacta caccgtggtg gaacagtacg
     1861 aacgctccga gggccgccac tccaccggcg gcatggacga gctgtacaag ggcaaaaaga
     1921 aaggccggac tcaaaaggag aagaaagctg ctcgtgctcg ttccaaaggg aaaaactcgg
     1981 atgaagaagc ccccaagact gcccagaaga agttaaagct cttataactt cgtataggat
     2041 actttatacg aagttatgga agcggagcga cgaattttag tctactaaaa caagcgggtg
     2101 atgtagaaga aaaccctgga cctatggcca agttgaccag tgccgttccg gtgctcaccg
     2161 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     2221 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     2281 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     2341 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     2401 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     2461 gcaactgcgt gcacttcgtg gccgaggagc aggacataac ttcgtatagg atactttata
     2521 cgaagttatt tggaagcgga gcgacgaatt ttagtctact gaaacaagcg ggagacgtgg
     2581 aggaaaaccc tggacctacc ggttgcggcc gcctcgagtc tagaactagt tgatcatgag
     2641 cccaataaag actgttaatt cctcatgcgt tgcctgccct tcctccattg ttgccctgga
     2701 atgtacagga cccaggggca gcagcagtcc aggtgccaca ggcagccctg ggacatagga
     2761 agctgggagc aaggaaaggg tcttagtcac tgcctcccga agttgcttga aagcactcgg
     2821 agaattgtgc aggtgtcatt tatctatgac caataggaag agcaaccagt tactatgagt
     2881 gaaagggagc cagaagactg attggagggc cctatcttgt gagtggggca tctgttggac
     2941 tttccacctg gtcatatact ctgcagctgt tagaatgtgc aagcacttgg ggacagcatg
     3001 agcttgctgt tgtacacagg gtatttctag aagcagaaat agactgggaa gatgcacaac
     3061 caaggggtta caggcatcgc ccatgctcct cacctgtatt ttgtaatcag aaataaattg
     3121 cttttaaaga aatctggcgt ctttgcactg tgtctgctgt ggaggcaggc ccctggcaaa
     3181 tggggggtga ggagcttgaa gagggtagaa tgggctgtgc taatatacag aatatatgta
     3241 acttgctata aattgaatga tcctttatag acaccgttta caaaccaaag acataaaatg
     3301 tggccagcag tgcctggtgc ttcctagtta atgtaaagct gtctcattct aattcagctg
     3361 caaagtatgg acccatgccc tgctgccagg ctgctgtagt cccggcggtc tgtagagact
     3421 agcattttgc aaatgataag cactagaata acagataacg ctaggcttta tgcactaaga
     3481 gaacccatta gatctgccag tctgaatccc agaaagggca ctgatacaga gcatttagta
     3541 ctaggtagca atttcataaa atgtccctgg tcaaagcttg ctggagcagg ccccgttccc
     3601 attttactga tgtctagcag tcgtgttcag taatacgtca tacccaggtc tccacgctaa
     3661 acagcctaaa cactaaaagt aaacatgctg tgaagtaaag gaagctgcgg tattttctcc
     3721 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     3781 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3841 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3901 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     3961 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4021 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4081 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4141 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4201 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4261 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4321 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4381 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4441 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4501 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4561 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4621 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4681 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4741 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4801 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4861 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4921 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4981 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5041 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5101 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5161 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5221 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5281 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5341 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5401 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5461 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5521 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5581 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5641 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5701 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5761 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5821 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     5881 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaaga
//
