LOCUS       sequence-502221-        8180 bp    DNA     circular SYN 10-MAR-2026
DEFINITION  Express Protein2PAM_NmeN4G.4 nuclease in human cells using a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8180)
  TITLE     Nayfach et al. Nature Biotechnology. 2025 (In Press)
REFERENCE   2  (bases 1 to 8180)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 10, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Protein2PAM_NmeN4G.4
FEATURES             Location/Qualifiers
     source          1..8180
                     /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"
     misc_feature    815..886
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     polyA_signal    1024..1145
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1061..1080)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1115..1134
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(1211..1228)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(1234..1947,1948..1950,1951..1953))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1237..1947,1948..1950,1951..1953))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1279..1300)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1628..1647
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1887..1908
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1954..2010)
                     /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             complement(2029..2094)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(2107..2127)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(5498..5700)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5527..5547)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5702..6005
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    6086..6134
                     /note="synthetic polyadenylation signal"
     terminator      6152..6198
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    6203..6274
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6426..6562)
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     CDS             complement(join(6575..7366,7367..7435))
                     /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     7198..7217
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
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 gctcacatga tttaaatgaa
      661 actcgtaatc ggagaacggg tgcaatagca aggaccagat cgaacttgac tgacgcaatt
      721 ctgagcgtag tcgaaaccca gacaagtcga tctcgccaag tagctgactt gactggttaa
      781 ttaagtttcg atagcccaag gtaaccaatg agattaggga gggaagaaag tttgatgaac
      841 tacttctgac ccctagtggt gtccagaaaa gaccattaaa ggaatgtctt gatcgaaatc
      901 gagagatcgc gataccgata gagatgggcc aataccacat ttgtagaggt tttacttgct
      961 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     1021 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1081 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     1141 tcttatcatg tctgtgagct gaaggtacgc tgtatctcag tcagtcaagc tagcagaggc
     1201 ctgcggatcc atggtgatgg tgatgatgac cggtcacttg tacagctcgt ccatgccgag
     1261 agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg
     1321 gtctttgctc agggcggact gggtgctcag gtagtggttg tcgggcagca gcacggggcc
     1381 gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat
     1441 gttgtggcgg atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata
     1501 gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt
     1561 gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc
     1621 ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc
     1681 ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa
     1741 gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc
     1801 ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc
     1861 ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac
     1921 cccggtgaac agctcctcgc ccttgctcac catcggtcca ggattctctt cgacatctcc
     1981 ggcttgtttc agcagagaga agtttgttgc gccgcttcca ccgcctccct tgtcatcgtc
     2041 atccttgtaa tcgatgtcat gatctttata atcaccgtca tggtctttgt agtcgcctcc
     2101 gctcgagact ttcctcttct tcttgggctc gaattcgctg ccgtcggcgg ttcttttccc
     2161 gcttccgcct ccgccggatc cccgcacggg tgggcgcttc ttcagcctac aaggtctgat
     2221 ctccttgccc agctcgtcga tctgatactt ctggaaggac agcagtgtct tcacgccgat
     2281 gccctccagg atgccgttct tgccgacctt gtgatccagg tcgtggatcc ggatattgat
     2341 ggcgcctgtg ctgcggtggc aggaggcgaa gtagccaaac atcctggcct tcttggttgt
     2401 cacttccacc aggtcattgg ggtgcagaga gaacttaaag ttgaaggaat cgtcgataac
     2461 ctgccaatcc tcctcgtcct tgccggccac aatggctcta tcaggcagga tgcccttggc
     2521 cacttgccag ctgtagatgg gcaccagata gtacttaccg cccttctcaa acacgtccac
     2581 ccgcaccatg gtagcattgt cggcgatgcc attgtggttg cggacccaca cgcctgtctt
     2641 ctgcacctgt tccaccctca cggccttcac ctgttgggtt ctgttgccgg ccttatcata
     2701 cttgtagaat ggctcggcaa aggccttggc aggatcgtcc ttgtgtgcct ccagccttgc
     2761 cttcagggcc tcatacagct ttggctcccg ctcgcgattc accatcttct ccagatcctt
     2821 cagcttcagc tgtgtcagag gcaccctcag cacagacacg ccctcgtcca gtctcttggc
     2881 ggacttcact gtctccatgt ggccctggcc gctcatcttc ctgttggggg ctctagacac
     2941 gaacagtggg gtcacgtact cgtgcactgc ctcgggcctg gagctcagct tctctgccag
     3001 cagtgttctc agcttctcca gggtatcggc ctcctcgaac tctggcttgc cgtcaggctt
     3061 gccaaacacc cggatcatca cttcctgggc aaagaactcc catggctgag gaaagtgtgt
     3121 cttctggtgc agcacctcgc ctgtctcctt atcgatggtc ttgccgtcga aggcattcat
     3181 ctccttatac cgcacaaagc gtgtgatctt ctgctgcatg gccacggtag aacatgccac
     3241 caccactgca tccagtgcgt ggtgcctgtc gttctctgcc ctcacctttc tcaggcccca
     3301 gaagcccctc agcagatttg tgatctggcc gttggaggca aacactctct tcttgccctt
     3361 gccggtcagc ctcattctat cggccacgaa ctggcacaga aaccgattca cgtagcgggt
     3421 gtcattcagg ttcctctcct tgaagccatc ctcgtcaaac ttctgcagca ggattctctg
     3481 cttcttggac cgagggaacc tgcttgtctc cacccttgcc ttaaactcct gccactcgcg
     3541 gctattgtcc ttgccgttga aatactcgta gggtgtctgg ttgcccttat tctggttctc
     3601 agagcccagc accagcacct tattgttgaa gctatcgtcc caggtccgag aaaaaggcag
     3661 ggcgtggtcg atctccacat agcccttctc attcaggcgg cccaggttga tctccttgcc
     3721 agaatacaga cacttgccgt gctgctgctc gtacagcctc agcttcagga tatccttgga
     3781 ctttggctcg cccacgaaat taggaaagta ctctctgaac ttggcggcgg ccttctccct
     3841 atcctttctg ttctcctcct gcctcttctc gatctccttt ctgtccttaa aggacttgcc
     3901 cacttccctg gctgtctcga tgtggattct ggcggggctg ccgtaccggc gcaccactcc
     3961 gttgatcact tttcttgcct gagacagggc gcgcagcacc acagggttcc tgatctcatc
     4021 ggcggggata gggggcaggt agattttctc ctctgtgttc ttcttgccat agtgatcgcc
     4081 gtagatttcg gcgcaggcct cgtcgtacct cttgccctgc tccatcagtg gcacgatcct
     4141 ccgcagggcc ttcaggctga tctgcacgaa cttatcaaag ctgatgtgct tcagcagggc
     4201 ctccaggatc tctggctgga tcctgtcctt cagtctgcct gtgatatcct cgtcggtctt
     4261 gaacaggcta aaggcggtgc cgatctcatc ctgcagctcg ggagacagat tcagtgggga
     4321 cttcttgtcc ttcaggccct ccttctccag ggccctgctg atggcgtgat aggccttcat
     4381 ctccatcagt gtagaggcct cggcgttatc cttgccgtat ctcaggccct taaagaaggc
     4441 ggtgtcctcc aggcccagca gcttccttgc ctgtgcatat gtcagcttgg acttgcggta
     4501 gggctcatcc atcagggtgg ccctctctgt gtcggtcagt gggcgctcgc ttccctgctc
     4561 caggatcctc agattgttca gctttgtcag ccagatgaac cgctcggctg tgtaggtatt
     4621 cttggcggcc ttgggctcgg ctggctcaaa ggtgcagtgt cccagcatct tctgcactgc
     4681 gtcgccgctc agggcgggcc gctgtgtcat cagcagtgtc tcgatgccct ccttcaggcc
     4741 gccagacacg tgtgggttgc cgaactcctt ctgcttctca aacagcagga tcagctcggc
     4801 ctgcagatcc ttccgggaga aggtgtggct atagtcagac cgctgattgc ggatgtggcc
     4861 gctctccttc tcgaacttgt tcagggccag ctcggctggt gtgcgaaagt cgccggtctg
     4921 cagggcgtgt gcatttcctg ccactccctt cagcagggcg cccagctcct tatcggctgt
     4981 ctcgccctcg ttctttctct gagacaggta gccccggtgc ttgatcaggt gcagcagcac
     5041 ggcggaccac tccagaggtg tcagcttgcg gtccagggcg gctgccctca gctgccaagg
     5101 ggtattgggc agggacttga tcaggccatt ctcatcgaag ttggcggcct gcagcacgcc
     5161 ctccctcttc agcagccggc gtgtcctcag cagtctgtgg gcccttctcc gggtcaggcg
     5221 cctcacggag cgtgccagtc tccgtgccat ggccaggctg tcgccggtct taggcacctc
     5281 tgccctctca aacacgcgca cgcccagatc gatcaggcgg atggggttct cctcctcgtc
     5341 gatttcgacc atagcccacc cgacggatgc gatgccgatg tccagtccca ggatgtaatt
     5401 gattgagttt ggcttgaatg ctgccatgcg gccgctggcg ggtgtgtcga gtggatggta
     5461 ggatcgacaa agatctgatc tgacggttca ctaaaccagc tctgcttata tagacctccc
     5521 accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa
     5581 agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg
     5641 gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca
     5701 gcatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg
     5761 tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg
     5821 tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca
     5881 cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg
     5941 acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg
     6001 taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactagatct
     6061 ggctacactc gatcagcagt tagataataa aatcgctatc catcgaagat ggatgtgtgt
     6121 tggttttttg tgtgtgtaac gcaacgattg atagcataac cccttggggc ctctaaacgg
     6181 gtcttgaggg gttttttgaa ttgagaggta gctgaagctg ctgttatgac cactagaggg
     6241 aagaagatac ctgtggagct aatggtccaa gatgtcggga gccgcgatcg ctgccaaaaa
     6301 ggtaagctga cgatagctga cgagattcga tgcccgacga gcaaatgagc gccacggctg
     6361 ttcgcgtaag ctcgaaacga cggagattcg atagagccag agtcgagttt aaacgattcg
     6421 ggatcttaag gataaatttc tggtaaggag gacacgtatg gaagtgggca agttggggaa
     6481 gccgtatccg ttgctgaatc tggcatatgt gggagtataa gacgcgcagc gtcgcatcag
     6541 gcattttttt ctgcgccaat gcacctagaa tcggttacca atgcttaatc agtgaggcac
     6601 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6661 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6721 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6781 gaagtggtcc tgcaacttta tccgcctcca tccagtctat caactgttgc cgggaagcta
     6841 gagtaagtag ttcgccagtt aatagtttac gcaaggttgt tgccattgct acaggcatcg
     6901 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6961 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccaatcg
     7021 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7081 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgaatac tcaaccaagt
     7141 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7201 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaagcgt tcttcggggc
     7261 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgcgcac
     7321 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7381 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatgcgaa
     7441 agcctcctgc tgatcctgtc tcttgataga tgtttgcaaa agctaggact cgattatacg
     7501 ttgaccgatc agactatcaa tatctaggtt taaacagatc ggtacgctgc cgtgaccagg
     7561 cttgagtaca atatctgacg attggacttc ctgggtccca agctggattc cgactggctg
     7621 actgccaagc aacgatatgg atcgtcgaga ctcgcactga gaggatcgtt ctcggtacac
     7681 caggcgcgcc ccgatttcga tattcctcgg atctcggata gatcgggatt gaggatgtag
     7741 gttgaacttc tctcgggtga tcccgatctc ggatattcgg gattggagta atacctggcg
     7801 ataggcgctg ccaagcaata gccaaggcgc aaggccggcc aagagacttg cgatagttcg
     7861 ttcgagaggt agcctgcaag atggatcgga cgaaaggttt gaagattttc cgacgagggc
     7921 cttggagagt tcggaaattt aaatgcaact atggatgaac gaaatagaca gatcgctgag
     7981 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     8041 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     8101 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     8161 gaaaagatca aaggatcttc
//
