LOCUS       sequence-325873-        7162 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 7162)
  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 7162)
  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: ACTB-PQR-RFPnols-PQR-loxP-Zeo-loxP
FEATURES             Location/Qualifiers
     source          1..7162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             1903..1959
                     /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             1960..2691
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     protein_bind    complement(2805..2838)
                     /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             2848..2904
                     /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             2905..3276
                     /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(3277..3310)
                     /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             3322..3378
                     /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    4653..4774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4690..4709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4744..4763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4781..5236)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4918..4939)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5130..5149
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5263..5367
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5369..5726
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5369..5560
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5392..5412)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5577..5712
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5761..6555
                     /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             5761..6555
                     /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(5815..5834)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6425..6444
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6743..6762)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6787..6834
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taataacact
      721 ggctcgtgtg acaaggccat gaggctggtg taaagcggcc ttggagtgtg tattaagtag
      781 gcgcacagta ggtctgaaca gactccccat cccaagaccc cagcacactt agccgtgttc
      841 tttgcacttt ctgcatgtcc cccgtctggc ctggctgtcc ccagtggctt ccccagtgtg
      901 acatggtgca tctctgcctt acagatcatg tttgagacct tcaacacccc agccatgtac
      961 gttgctatcc aggctgtgct atccctgtac gcctctggcc gtaccactgg catcgtgatg
     1021 gactccggtg acggggtcac ccacactgtg cccatctacg aggggtatgc cctcccccat
     1081 gccatcctgc gtctggacct ggctggccgg gacctgactg actacctcat gaagatcctc
     1141 accgagcgcg gctacagctt caccaccacg gccgagcggg aaatcgtgcg tgacattaag
     1201 gagaagctgt gctacgtcgc cctggacttc gagcaagaga tggccacggc tgcttccagc
     1261 tcctccctgg agaagagcta cgagctgcct gacggccagg tcatcaccat tggcaatgag
     1321 cggttccgct gccctgaggc actcttccag ccttcctccc tgggtgagtg gagactgtct
     1381 cccggctctg cctgacatga gggttacccc tcggggctgt gctgtggaag ctaagtcctg
     1441 ccctcatttc cctctcaggc atggagtcct gtggcatcca cgaaactacc ttcaactcca
     1501 tcatgaagtg tgacgtggac atccgcaaag acctgtacgc caacacagtg ctgtctggcg
     1561 gcaccaccat gtaccctggc attgccgaca ggatgcagaa ggagatcact gccctggcac
     1621 ccagcacaat gaagatcaag gtgggtgtct ttcctgcctg agctgacctg ggcaggtcag
     1681 ctgtggggtc ctgtggtgtg tggggagctg tcacatccag ggtcctcact gcctgtcccc
     1741 ttccctcctc agatcattgc tcctcctgag cgcaagtact ccgtgtggat cggcggctcc
     1801 atcctggcct cgctgtccac cttccagcag atgtggatca gcaagcagga gtatgacgag
     1861 tccggcccct ccatcgtcca ccgcaaatgc ttcggaagcg gagcgacgaa ttttagtcta
     1921 ctaaaacaag cgggtgatgt agaagaaaac cctggaccta tggtgtctaa gggcgaagag
     1981 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgaa caaccaccac
     2041 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc
     2101 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac cagcttcatg
     2161 tacggcagca gaaccttcat caaccacacc cagggcatcc ccgatttctt taagcagtcc
     2221 ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc
     2281 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg
     2341 gtgaacttcc catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccaac
     2401 accgagatgc tgtaccccgc tgacggcggc ctggaaggca gaaccgacat ggccctgaag
     2461 ctcgtgggcg ggggccacct gatctgcaac ttcaagacca catacagatc caagaaaccc
     2521 gctaagaacc tcaagatgcc cggcgtctac tatgtggacc acagactgga aagaatcaag
     2581 gaggccgaca aagagaccta cgtcgagcag cacgaggtgg ctgtggccag atactgcgac
     2641 ctccctagca aactggggca caaacttaat ggcatggacg agctgtacaa gggcaaaaag
     2701 aaaggccgga ctcaaaagga gaagaaagct gctcgtgctc gttccaaagg gaaaaactcg
     2761 gatgaagaag cccccaagac tgcccagaag aagttaaagc tcttataact tcgtatagga
     2821 tactttatac gaagttatgg aagcggagcg acgaatttta gtctactaaa acaagcgggt
     2881 gatgtagaag aaaaccctgg acctatggcc aagttgacca gtgccgttcc ggtgctcacc
     2941 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac
     3001 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg
     3061 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac
     3121 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg
     3181 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc
     3241 ggcaactgcg tgcacttcgt ggccgaggag caggacataa cttcgtatag gatactttat
     3301 acgaagttat ttggaagcgg agcgacgaat tttagtctac tgaaacaagc gggagacgtg
     3361 gaggaaaacc ctggacctac cggttgcggc cgcctcgagt ctagaactag ttgatcatag
     3421 gcggactatg acttagttgc gttacaccct ttcttgacaa aacctaactt gcgcagaaaa
     3481 caagatgaga ttggcatggc tttatttgtt ttttttgttt tgttttggtt tttttttttt
     3541 ttttggcttg actcaggatt taaaaactgg aacggtgaag gtgacagcag tcggttggag
     3601 cgagcatccc ccaaagttca caatgtggcc gaggactttg attgcacatt gttgtttttt
     3661 taatagtcat tccaaatatg agatgcattg ttacaggaag tcccttgcca tcctaaaagc
     3721 caccccactt ctctctaagg agaatggccc agtcctctcc caagtccaca caggggaggt
     3781 gatagcattg ctttcgtgta aattatgtaa tgcaaaattt ttttaatctt cgccttaata
     3841 cttttttatt ttgttttatt ttgaatgatg agccttcgtg cccccccttc cccctttttt
     3901 gtcccccaac ttgagatgta tgaaggcttt tggtctccct gggagtgggt ggaggcagcc
     3961 agggcttacc tgtacactga cttgagacca gttgaataaa agtgcacacc ttaaaaatga
     4021 ggccaagtgt gactttgtgg tgtggctggg ttgggggcag cagagggtga accctgcagg
     4081 agggtgaacc ctgcaaaagg gtggggcagt gggggccaac ttgtcctcac ccagagtgca
     4141 ggtgtgtgga gatccctcct gccttgacat tgagcagcct tagagggtgg gggaggctca
     4201 ggggtcaggt ctctgttcct gcttattggg gagttcctgg cctggccctt ctatgtctcc
     4261 ccaggtaccc cagtttttct gggttcaccc agagtgcaga tgcttgagga ggtgggaagg
     4321 gactatttgg gggtgtctgg ctcaggtgcc atgcctcact ggggctggtt ggcacctgca
     4381 tttcctggga gtggggctgt ctcagggtag ctgggcacgg tgttcccttg agtgggggtg
     4441 tagtgagtgt tcctagctgc cacgcctttg ccttcaccta tgggatcgtg gctgtcggcc
     4501 ttgagggtca gcctggccca ggctcccata ggcttaggag aggccgcaat tcctacctgt
     4561 tcatccagac agagggggac ctggaatcaa agtcaagttg gggtaggggc cccctgaacc
     4621 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     4681 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4741 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     4801 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4861 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     4921 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4981 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5041 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     5101 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     5161 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     5221 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5281 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5341 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     5401 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     5461 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     5521 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     5581 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5641 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     5701 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     5761 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     5821 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     5881 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     5941 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     6001 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     6061 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     6121 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     6181 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     6241 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     6301 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     6361 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     6421 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     6481 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     6541 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     6601 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     6661 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     6721 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     6781 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     6841 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     6901 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     6961 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     7021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     7081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     7141 tagaaaagat caaaggatct tc
//
