LOCUS       sequence-417248-        6645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Universal Antibiotic Screening Reporter for adenine and cytosine 
            base editors. Key Construct: CMV-PuroR*(ATAACG)-eGFP-hU6-gRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6645)
  AUTHORS   Ma L, Xing J, Li Q, Zhang Z, Xu K
  TITLE     Development of a universal antibiotic resistance screening reporter 
            for improving efficiency of cytosine and adenine base editing.
  JOURNAL   J Biol Chem. 2022 Jul;298(7):102103. doi: 10.1016/j.jbc.2022.102103.
            Epub 2022 Jun 6.
  PUBMED    35671823
REFERENCE   2  (bases 1 to 6645)
  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: "J Biol 
            Chem."; date: "2022-07"; volume: "298(7):102103. doi"; pages: " 
            10.1016/j.jbc.2022.102103. Epub 2022 Jun 6"
COMMENT     Sequence Label: pACBE-ASR (pXK-607)
FEATURES             Location/Qualifiers
     source          1..6645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        24..411
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        412..615
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     566..586
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             654..1247
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TECKPRVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1147..1167
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1272..1325
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1326..1328,1329..2042)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIDDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1368..1389)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1629..1648)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1976..1997
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2160..2281
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2197..2216)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2251..2270
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2288..2536
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2288..2308
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2459..2478
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2557..2632
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   2996..3030
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3521..3537)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3521..3537)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3534..3556)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3545..3561
                     /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(3569..3575,3576..3593,3594..3599))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3614..3635
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3752..3769)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3923..4511)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4003..4022)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4682..5473,5474..5542))
                     /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     5305..5324
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5543..5647)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5673..6128)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5810..5831)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6022..6041
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6255..6277
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6269..6286
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6270..6286
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   6416..6478
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 acttttatcg aattcacggc ctcgacattg attattgact agagcattag ttattaatag
       61 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      121 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      181 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      241 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      301 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      361 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      421 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      481 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      541 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      601 tatataagca gagctggttt agtgaaccgt cagatcccgc taccgccaat ataacggaat
      661 gcaagcccag ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
      721 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
      781 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
      841 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
      901 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
      961 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     1021 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     1081 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     1141 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     1201 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccatg cgagctaccg
     1261 gtggcagtgg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg
     1321 gcccagtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1381 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1441 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1501 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1561 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1621 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1681 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1741 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1801 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1861 ccgaccacta ccagcagaac acccccatcg acgacggccc cgtgctgctg cccgacaacc
     1921 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1981 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     2041 aatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     2101 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     2161 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2221 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2281 aaagcttgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     2341 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     2401 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     2461 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     2521 gaaaggacga aacaccatat aacggaatgc aagcccgttt tagagctaga aatagcaagt
     2581 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
     2641 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
     2701 tctgcagaca aatggctcta ggatatctat aacaagaaaa tatatatata ataagttatc
     2761 acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac
     2821 gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac
     2881 acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa
     2941 atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc
     3001 gtcaatttta cgcagactat ctttctaggg ttaatctagc tgcatcagga tcatatcgtc
     3061 gggtcttttt tccggctcag tcatcgccca agctggcgct atctgggcat cggggaggaa
     3121 gaagcccgtg ccttttcccg cgaggttgaa gcggcatgga aagagtttgc cgaggatgac
     3181 tgctgctgca ttgacgttga gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg
     3241 gccatgcacg cctttaacgg tgaactgttc gttcaggcca cctgggatac cagttcgtcg
     3301 cggcttttcc ggacacagtt ccggatggtc agcccgaagc gcatcagcaa cccgaacaat
     3361 accggcgaca gccggaactg ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg
     3421 ggatattacg tcagcgagga cgggtatcct ggctggatgc cgcagaaatg gacatggata
     3481 ccccgtgagt tacccggcgg gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3541 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3601 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3661 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3721 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3781 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3841 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3901 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3961 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4021 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4081 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4141 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4201 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4261 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4321 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4381 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4441 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4501 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4561 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4621 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4681 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4741 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4801 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4861 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4921 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4981 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5041 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5101 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5161 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5221 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5281 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5341 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5401 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5461 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5521 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5581 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5641 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     5701 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     5761 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     5821 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     5881 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     5941 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     6001 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     6061 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     6121 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     6181 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     6241 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcctcgtt
     6301 cattcacgtt tttgaacccg tggaggacgg gcagactcgc ggtgcaaatg tgttttacag
     6361 cgtgatggag cagatgaaga tgctcgacac gctgcagaac acgcagctag attaacccta
     6421 gaaagataat catattgtga cgtacgttaa agataatcat gtgtaaaatt gacgcatgtg
     6481 ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta agttttatta
     6541 tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt
     6601 attaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaa
//
