LOCUS       sequence-370112-        6900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell line generation via recombination-mediated cassette exchange 
            (RMCE) and stable expression of EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6900)
  AUTHORS   Villasenor R, Pfaendler R, Ambrosi C, Butz S, Giuliani S, Bryan E, 
            Sheahan TW, Gable AL, Schmolka N, Manzo M, Wirz J, Feller C, von 
            Mering C, Aebersold R, Voigt P, Baubec T
  TITLE     ChromID identifies the protein interactome at chromatin marks.
  JOURNAL   Nat Biotechnol. 2020 Mar 2. pii: 10.1038/s41587-020-0434-2. doi: 
            10.1038/s41587-020-0434-2.
  PUBMED    32123383
REFERENCE   2  (bases 1 to 6900)
  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: "Nat 
            Biotechnol. 2020 Mar 2. pii: 10.1038/s41587-020-0434-2. doi: 
            10.1038/s41587-020-0434-2."
COMMENT     Sequence Label: pv6_EGFP
FEATURES             Location/Qualifiers
     source          1..6900
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    409..442
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     enhancer        447..826
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1104..2120
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2044..2066
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2128..2147
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2188..2232
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2230..2250
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2326..2346
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2374..2376,2377..3090)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2374..2376,2377..3087)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2416..2437)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2677..2696)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3024..3045
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3213..3675
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3269..3286)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3496..3515
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3689..4288
                     /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(3689..3708)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4185..4205
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4457..4578
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4494..4513)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4548..4567
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4612..4645)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     primer_bind     complement(4679..4695)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4679..4695)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4692..4714)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4703..4719
                     /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)."
     promoter        complement(join(4727..4733,4734..4751,4752..4757))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4772..4793
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4910..4927)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5081..5669)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5161..5180)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5840..6631,6632..6700))
                     /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     6463..6482
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6701..6805)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6873..6891
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ctcgagccat aacttcgtat
      421 aatgtatact atacgaagtt atatcggaca ttgattattg actagttatt aatagtaatc
      481 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      541 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      601 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      661 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      721 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      781 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      841 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      901 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggsgc gcgccrggsg
      961 gggsggggsg gggsgrgggg sggggsgggg sgrggcggag aggtgcggcg gcagccaatc
     1021 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     1081 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     1141 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1201 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
     1261 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
     1321 ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg
     1381 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg
     1441 tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga
     1501 acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg
     1561 tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg
     1621 gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc
     1681 aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc
     1741 gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt
     1801 tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga
     1861 aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg
     1921 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc
     1981 ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg
     2041 cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg
     2101 ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt
     2161 ttggcaaaga attctagcat ggctggtggc ctgaatgaca tctttgaggc ccagaagatc
     2221 gagtggcatg agaacctgta cttccaggga gtcgacgaag agtctggttc tgtttctggt
     2281 ggtgatatcg gtggcggagg ctctggaggt ggaggctccg caccccccaa aaagaaacgc
     2341 aaggttggat accgggatcc accggtcgcc accgtgagca agggcgagga gctgttcacc
     2401 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2461 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     2521 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     2581 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2641 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2701 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2761 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2821 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2881 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2941 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     3001 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     3061 actctcggca tggacgagct gtacaagtaa agcgagctcg acagatctag gagcggttaa
     3121 cgaggtagcg gccgccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     3181 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     3241 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     3301 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     3361 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     3421 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     3481 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     3541 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     3601 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     3661 aaaacacgat gataatatgg ccacaaccat gaccgagtac aagcccacgg tgcgcctcgc
     3721 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     3781 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     3841 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     3901 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     3961 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     4021 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     4081 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     4141 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     4201 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     4261 gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga
     4321 aaggagcgca gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4381 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4441 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4501 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4561 actcatcaat gtatcttatc atgtctgtat accgtggatc cggccggccc cataacttcg
     4621 tatagtatac attatacgaa gttatcccct gcaggcatgc aagcttggcg taatcatggt
     4681 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4741 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4801 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4861 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4921 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4981 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5041 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5101 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5161 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5221 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5281 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5341 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5401 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5461 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5521 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5581 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5641 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5701 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5761 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5821 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5881 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5941 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6001 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6061 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6121 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6181 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6241 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6301 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6361 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6421 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6481 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6541 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6601 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6661 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6721 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6781 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     6841 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
