LOCUS       sequence-346305-        6817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expressing retron Ec73ncRNA-gRNA chimeric RNA (rgRNA) 
            targeting HEK3 site driven by EF1alpha promoter and 
            EGFP(L138S)-P2A-mCherry driven by CMV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6817)
  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 6817)
  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_HH-HEK3 spacer-gRNA scaffold-EC73-5'-don...
FEATURES             Location/Qualifiers
     source          1..6817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   48..110
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        413..1591
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          644..1582
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1539..1559
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     ncRNA           1608..1644
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        1665..1740
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           1991..2058
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(2068..2085)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2074..2281
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        2348..2651
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2652..2855
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2806..2826
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2878..2887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2884..2886,2887..2889,2890..3600)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNISGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2929..2950)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3190..3209)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3537..3558
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3601..3657
                     /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      3664..3673
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3670..4380
                     /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(3815..3833)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    4397..4621
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4978..5012
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        5043..5147
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5148..5216,5217..6008)
                     /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(5366..5385)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6179..6767
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6668..6687
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgctcacatg ttctttcctg cgttatcccc tgattctctc gagttaaccc tagaaagata
       61 atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg tgttttatcg
      121 atctgtatat cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca
      181 cttacatact aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa
      241 aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttaaacat tctctctttt
      301 acaaaaataa acttattttg tactttaaaa acagtcatgt tgtattataa aataagtaat
      361 tagcttaact tatacataat agaaacaaat tatacttatt aattaatcta gaggctccgg
      421 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
      481 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
      541 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
      601 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
      661 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
      721 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
      781 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
      841 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
      901 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
      961 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
     1021 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     1081 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     1141 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     1201 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     1261 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     1321 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     1381 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     1441 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     1501 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     1561 aagttttttt cttccatttc aggtgtcgtg aacgcgtagt ctgggccctg atgagtccgt
     1621 gaggacgaaa cgagtaagct cgtcggccca gactgagcac gtgagtttta gagctagaaa
     1681 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     1741 gcagagccaa acctagcatt ttatgggtta atagcccatc gggccatgag tcatggtttc
     1801 gcctagtatt ttagctatgc ccgtcgttac tagtgcttct ccagccctgg cctgggtcaa
     1861 tccttggggc ccagactgag cactagtgat ggcagaggaa aggaagccct gcttcctcca
     1921 gagggcgtcg caggacagct tttcctagac aggggcccta ggatcgacgt gctcaagtag
     1981 gtttggctct ggccggcatg gtcccagcct cctcgctggc gccggctggg caacatgctt
     2041 cggcatggcg aatgggacac gcgtcagcct cgactgtgcc ttctagttgc cagccatctg
     2101 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2161 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2221 gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg
     2281 aaagctttag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     2341 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     2401 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     2461 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     2521 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     2581 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     2641 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     2701 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     2761 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     2821 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgcc
     2881 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2941 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3001 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3061 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     3121 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     3181 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3241 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catttcaggg
     3301 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     3361 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     3421 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     3481 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     3541 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3601 gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctgct
     3661 agcgccacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
     3721 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     3781 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     3841 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     3901 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     3961 tgggagcgcg tgatgaactt cgatgacggc ggcgtggtga ccgtgaccca ggactcctcc
     4021 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     4081 ggccccgtaa tgcagaagaa aaccatgggc tgggaggcct cctccgagcg gatgtacccc
     4141 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     4201 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     4261 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     4321 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtaa
     4381 ctcttggccc ggatccctgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4441 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4501 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     4561 agcaaggggg aggattggga aaacaatagc aggcatgctg gggatgcggt gggctctatg
     4621 ggtcgacggc gcgccctaga cgaagcagct ccagcctaca caatcgctca agacgtgtaa
     4681 tgcttttatt atatattagt cacgatatct ataacaagaa aatatatata taataagtta
     4741 tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat cttaaaagtc
     4801 acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc aaaatcagtg
     4861 acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact gagatgtcct
     4921 aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc aagaatgcat
     4981 gcgtcaattt tacgcagact atctttctag ggttaaggat ccgcactttt cggggaaatg
     5041 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5101 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5161 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5221 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5281 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5341 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5401 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5461 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5521 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5581 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5641 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5701 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5761 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5821 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5881 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5941 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6001 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6061 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6121 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6181 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6241 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6301 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6361 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6421 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6481 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6541 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6601 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6661 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6721 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6781 cggccttttt acggttcctg gccttttgct ggccttt
//
