LOCUS       sequence-346306-        6656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for constructing retron Ec73ncRNA-gRNA chimeric RNA (Ec73 
            rgRNA), inserting spacer sequence by BbsI digestion and inserting 
            donor sequence by EcoRI digestion. EGFP(L202S)-P2A-mCherry driven by
            CMV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6656)
  AUTHORS   Kong X, Wang Z, Zhang R, Wang X, Zhou Y, Shi L, Yang H
  TITLE     Precise genome editing without exogenous donor DNA via retron 
            editing system in human cells.
  JOURNAL   Protein Cell. 2021 Aug 17. pii: 10.1007/s13238-021-00862-7. doi: 
            10.1007/s13238-021-00862-7.
  PUBMED    34403072
REFERENCE   2  (bases 1 to 6656)
  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: "Protein 
            Cell. 2021 Aug 17. pii: 10.1007/s13238-021-00862-7. doi: 
            10.1007/s13238-021-00862-7."
COMMENT     Sequence Label: pEF1a_BbsI-gRNA
            scaffold-EC73-5'-EcoRI-EC73-3'...
FEATURES             Location/Qualifiers
     source          1..6656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..64
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        367..1545
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          598..1536
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1493..1513
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_RNA        1570..1645
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           1784..1851
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(1861..1878)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1867..2074
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        2141..2444
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2445..2648
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2599..2619
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2671..2680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2677..2679,2680..2682,2683..3393)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYSSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2722..2743)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2983..3002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3330..3351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3394..3450
                     /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"
     regulatory      3457..3466
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3463..4173
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     DDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3608..3626)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    4190..4414
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4771..4805
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        4836..4940
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4941..5009,5010..5801)
                     /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     complement(5159..5178)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5972..6560
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6461..6480
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg
       61 catgtgtttt atcgatctgt atatcgaggt ttatttatta atttgaatag atattaagtt
      121 ttattatatt tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat
      181 ttatttatta aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttaa
      241 acattctctc ttttacaaaa ataaacttat tttgtacttt aaaaacagtc atgttgtatt
      301 ataaaataag taattagctt aacttataca taatagaaac aaattatact tattaattaa
      361 tctagaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
      421 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
      481 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
      541 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
      601 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
      661 tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt
      721 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag
      781 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc
      841 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt
      901 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt
      961 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc
     1021 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg
     1081 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg
     1141 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa
     1201 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc
     1261 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac
     1321 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat
     1381 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg
     1441 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct
     1501 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaacgcg tgggtcttcg
     1561 agaagacctg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     1621 tgaaaaagtg gcaccgagtc ggtgcgcaga gccaaaccta gcattttatg ggttaatagc
     1681 ccatcgggcc atgagtcatg gtttcgccta gtattttagc tatgcccgtc gttgaattcc
     1741 agttcgctgg aattcatcga cgtgctcaag taggtttggc tctggccggc atggtcccag
     1801 cctcctcgct ggcgccggct gggcaacatg cttcggcatg gcgaatggga cacgcgtcag
     1861 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1921 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1981 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2041 aggattggga agagaatagc aggcatgctg gggaaagctt tagttattaa tagtaatcaa
     2101 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     2161 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2221 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2281 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2341 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2401 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2461 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     2521 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2581 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2641 gcagagctgg tttagtgaac cgtcagatcc gccaccatgg tgagcaaggg cgaggagctg
     2701 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2761 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2821 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2881 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2941 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     3001 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3061 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     3121 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     3181 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     3241 atcggcgacg gccccgtgct gctgcccgac aaccactatt caagtaccca gtcggccctg
     3301 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     3361 gggatcactc tcggcatgga cgagctgtac aaggctacta acttcagcct gctgaagcag
     3421 gctggagacg tggaggagaa ccctggacct gctagcgcca ccatggtgag caagggcgag
     3481 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     3541 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     3601 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     3661 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     3721 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgatgac
     3781 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     3841 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaaaaccatg
     3901 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     3961 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     4021 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     4081 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     4141 tccaccggcg gcatggacga gctgtacaag taactcttgg cccggatccc tgtgccttct
     4201 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     4261 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4321 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaaaacaat
     4381 agcaggcatg ctggggatgc ggtgggctct atgggtcgac ggcgcgccct agacgaagca
     4441 gctccagcct acacaatcgc tcaagacgtg taatgctttt attatatatt agtcacgata
     4501 tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa
     4561 tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt
     4621 gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct
     4681 cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat
     4741 ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc
     4801 tagggttaag gatccgcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4861 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4921 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4981 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5041 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5101 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5161 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5221 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5281 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5341 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5401 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5461 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5521 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5581 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5641 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5701 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5761 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5821 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5881 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5941 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6001 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6061 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6121 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6181 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6241 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6301 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6361 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6421 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6481 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6541 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6601 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctctc gagtta
//
