LOCUS       sequence-239710-        6960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To make a stable human recombinant cell line; see right thumbnail 
            map image for PQR annotations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6960)
  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 6960)
  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-RFPnols-PQR-loxP-Zeo-loxP
FEATURES             Location/Qualifiers
     source          1..6960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1140..1196
                     /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             1197..1928
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     protein_bind    complement(2042..2075)
                     /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             2085..2141
                     /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             2142..2519
                     /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
                     EFALRDPAGNCVHFVAEEQDL"
     protein_bind    complement(2516..2549)
                     /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             2559..2615
                     /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"
     polyA_signal    3807..3928
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3844..3863)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3898..3917
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3935..4390)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4072..4093)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4284..4303
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4417..4521
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4523..4880
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4523..4714
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4546..4566)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4731..4866
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4915..5709
                     /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             4915..5709
                     /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(4969..4988)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5579..5598
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5897..5916)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5941..5988
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6317..6905
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6806..6825
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcatt
       61 agttattaat tctgactggg cctgctatct gtcacccaac aggtatgctg ccccacaaaa
      121 ccaagcgagg ccaggccgct ctggaccgtc tcaaggtgtt tgacggcatc ccaccgccct
      181 acgacaaggt gagctatgcc aaaccccaca ggcagcggcc ttacctgtgg cgtccatgat
      241 gttccgcaac tacctacatt gtttgatcct catgaaagca gcactggctg agacgccagt
      301 ccagccgacc tccttccctg tctgtccctc ccgggctctt aagcccctct ctttctctaa
      361 cagaaaaagc ggatggtggt tcctgctgcc ctcaaggtcg tgcgtctgaa gcctacaaga
      421 aaggtgagtc ccagcttacg ctgcaccatc tacttgggag atttcaggcc tgctgaggga
      481 cctggggacc tggagcctgg cagatgatgt ccttatctca cgatggtctg cggatgtccc
      541 tgtgggaatg gcgacaatgc caatggctta gctgatgcca ggaggcttgg gtgggtgctt
      601 ttctaacagg cctgcagaga acagttgcat tatgatatgc ccagctgtca gtcacctccc
      661 agctctcaac agctccggct cttcagggtg tgggggctta gatatcctta caacttcatt
      721 tgttcacccc ccccccccgc agtttgccta tctggggcgc ctggctcacg aggttggctg
      781 gaagtaccag gcagtgacag ccaccctgga ggagaagagg aaagagaaag ccaagatcca
      841 ctaccggaag aagaaacagc tcatggtgag gccaggggct ggtgctgagg ggggcatctc
      901 actcctggac aggcctggca ggtgccttgc tcacagagta ctcttaactg gcaaaggacc
      961 agccggggtt ggggtgggat gcagtccatg taatgagggc aatgcaaccc ctcctgacca
     1021 ccaccacctg cacttattct tggcagaggc tacggaaaca ggccgagaag aacgtggaga
     1081 agaaaattga caaatacaca gaggtcctca agacccacgg actcctggtc ggaagcggag
     1141 cgacgaattt tagtctacta aaacaagcgg gtgatgtaga agaaaaccct ggacctatgg
     1201 tgtctaaggg cgaagagctg attaaggaga acatgcacat gaagctgtac atggagggca
     1261 ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
     1321 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
     1381 tggctaccag cttcatgtac ggcagcagaa ccttcatcaa ccacacccag ggcatccccg
     1441 atttctttaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag
     1501 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
     1561 acgtcaagat cagaggggtg aacttcccat ccaacggccc tgtgatgcag aagaaaacac
     1621 tcggctggga ggccaacacc gagatgctgt accccgctga cggcggcctg gaaggcagaa
     1681 ccgacatggc cctgaagctc gtgggcgggg gccacctgat ctgcaacttc aagaccacat
     1741 acagatccaa gaaacccgct aagaacctca agatgcccgg cgtctactat gtggaccaca
     1801 gactggaaag aatcaaggag gccgacaaag agacctacgt cgagcagcac gaggtggctg
     1861 tggccagata ctgcgacctc cctagcaaac tggggcacaa acttaatggc atggacgagc
     1921 tgtacaaggg caaaaagaaa ggccggactc aaaaggagaa gaaagctgct cgtgctcgtt
     1981 ccaaagggaa aaactcggat gaagaagccc ccaagactgc ccagaagaag ttaaagctct
     2041 tataacttcg tataggatac tttatacgaa gttatggaag cggagcgacg aattttagtc
     2101 tactaaaaca agcgggtgat gtagaagaaa accctggacc tatggccaag ttgaccagtg
     2161 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     2221 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     2281 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     2341 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     2401 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     2461 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gacttataac
     2521 ttcgtatagg atactttata cgaagttatg gaagcggagc gacgaatttt agtctactga
     2581 aacaagcggg agacgtggag gaaaaccctg gacctaccgg ttgcggccgc ctcgagtcta
     2641 gaactagttg atcatgagcc caataaagac tgttaattcc tcatgcgttg cctgcccttc
     2701 ctccattgtt gccctggaat gtacgggacc caggggcagc agcagtccag gtgccacagg
     2761 cagccctggg acataggaag ctgggagcaa ggaaagggtc ttagtcactg cctcccgaag
     2821 ttgcttgaaa gcactcggag aattgtgcag gtgtcattta tctatgacca ataggaagag
     2881 caaccagtta ctatgagtga aagggagcca gaagactgat tggagggccc tatcttgtga
     2941 gtggggcatc tgttggactt tccacctggt catatactct gcagctgtta gaatgtgcaa
     3001 gcacttgggg acagcatgag cttgctgttg tacacagggt atttctagaa gcagaaatag
     3061 actgggaaga tgcacaacca aggggttaca ggcatcgccc atgctcctca cctgtatttt
     3121 gtaatcagaa ataaattgct tttaaagaaa tctggcgtct ttgcactgtg tctgctgtgg
     3181 aggcaggccc ctggcaaatg gggggtgagg agcttgaaga gggtagaatg ggctgtgcta
     3241 atatacagaa tatatgtaac ttgctataaa ttgaatgatc ctttatagac accgtttaca
     3301 aaccaaagac ataaaatgtg gccagcagtg cctggtgctt cctagttaat gtaaagctgt
     3361 ctcattctaa ttcagctgca aagtatggac ccatgccctg ctgccaggct gctgtagtcc
     3421 cggcggtctg tagagactag cattttgcaa atgataagca ctagaataac agataacgct
     3481 aggctttatg cactaagaga acccattaga tctgccagtc tgaatcccag aaagggcact
     3541 gatacagagc atttagtact aggtagcaat ttcataaaat gtccctggtc aaagcttgct
     3601 ggagcaggcc ccgttcccat tttactgatg tctagcagtc gtgttcagta atacgtcata
     3661 cccaggtctc cacgctaaac agcctaaaca ctaaaagtaa acatgctgtg aagtaaagga
     3721 agctagattc atgctgtcct tgcggttgga tgccaagatt cagccccctg aacctgaaac
     3781 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3841 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3901 gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta
     3961 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     4021 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     4081 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     4141 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4201 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4261 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4321 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4381 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4441 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4501 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     4561 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4621 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4681 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4741 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4801 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4861 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4921 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4981 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     5041 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     5101 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     5161 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     5221 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     5281 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5341 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5401 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     5461 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5521 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5581 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5641 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5701 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5761 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5821 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5881 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     5941 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     6001 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     6061 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     6121 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     6181 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6241 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6301 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6361 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6421 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6481 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6541 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6601 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6661 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6721 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6781 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6841 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6901 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
//
