LOCUS       sequence-346304-        6818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expressing retron Ec73ncRNA-gRNA chimeric RNA (rgRNA) 
            targeting EMX1 driven by EF1alpha promoter and and 
            EGFP(L138S)-P2A-mCherry driven by CMV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6818)
  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 6818)
  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-EMX1 spacer-gRNA scaffold-EC73-5'-don...
FEATURES             Location/Qualifiers
     source          1..6818
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   82..144
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        447..1625
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          678..1616
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1573..1593
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     ncRNA           1642..1678
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        1699..1774
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           2026..2093
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(2103..2120)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2109..2316
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        2383..2686
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2687..2890
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2841..2861
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2913..2922
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2919..2921,2922..2924,2925..3635)
                     /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(2964..2985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3225..3244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3572..3593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3636..3692
                     /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      3699..3708
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3705..4415
                     /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(3850..3868)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    4432..4656
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5013..5047
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        5078..5182
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5183..5251,5252..6043)
                     /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(5401..5420)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6214..6802
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6703..6722
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
       61 cccctgattc tctcgagtta accctagaaa gataatcata ttgtgacgta cgttaaagat
      121 aatcatgcgt aaaattgacg catgtgtttt atcgatctgt atatcgaggt ttatttatta
      181 atttgaatag atattaagtt ttattatatt tacacttaca tactaataat aaattcaaca
      241 aacaatttat ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa atttcttcta
      301 taaagtaaca aaacttttaa acattctctc ttttacaaaa ataaacttat tttgtacttt
      361 aaaaacagtc atgttgtatt ataaaataag taattagctt aacttataca taatagaaac
      421 aaattatact tattaattaa tctagaggct ccggtgcccg tcagtgggca gagcgcacat
      481 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
      541 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
      601 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
      661 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg
      721 gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc
      781 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt
      841 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt
      901 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat
      961 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc
     1021 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca
     1081 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
     1141 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg
     1201 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc
     1261 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac
     1321 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt
     1381 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag
     1441 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag
     1501 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat
     1561 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt
     1621 cgtgaacgcg tgctcggact cctgatgagt ccgtgaggac gaaacgagta agctcgtcga
     1681 gtccgagcag aagaagaagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     1741 ttatcaactt gaaaaagtgg caccgagtcg gtgcgcagag ccaaacctag cattttatgg
     1801 gttaatagcc catcgggcca tgagtcatgg tttcgcctag tattttagct atgcccgtcg
     1861 ttactagtac aaacggcaga agctggagga ggaagggcct gagtccgagc agaagaagct
     1921 taagggctcc catcacatca accggtggcg cattgccacg aagcaggcca atggggagga
     1981 catcgatgtc acctaggatc gacgtgctca agtaggtttg gctctggccg gcatggtccc
     2041 agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca tggcgaatgg gacacgcgtc
     2101 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2161 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2221 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2281 ggaggattgg gaagagaata gcaggcatgc tggggaaagc tttagttatt aatagtaatc
     2341 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     2401 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     2461 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     2521 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     2581 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     2641 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     2701 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     2761 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     2821 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     2881 aagcagagct ggtttagtga accgtcagat ccgccaccat ggtgagcaag ggcgaggagc
     2941 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     3001 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     3061 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     3121 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     3181 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     3241 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     3301 gcatcgactt caaggaggac ggcaacattt cagggcacaa gctggagtac aactacaaca
     3361 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     3421 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     3481 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     3541 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     3601 ccgggatcac tctcggcatg gacgagctgt acaaggctac taacttcagc ctgctgaagc
     3661 aggctggaga cgtggaggag aaccctggac ctgctagcgc caccatggtg agcaagggcg
     3721 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
     3781 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     3841 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     3901 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     3961 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgatg
     4021 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     4081 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaaaacca
     4141 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     4201 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     4261 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     4321 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     4381 actccaccgg cggcatggac gagctgtaca agtaactctt ggcccggatc cctgtgcctt
     4441 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     4501 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     4561 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaaaaca
     4621 atagcaggca tgctggggat gcggtgggct ctatgggtcg acggcgcgcc ctagacgaag
     4681 cagctccagc ctacacaatc gctcaagacg tgtaatgctt ttattatata ttagtcacga
     4741 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
     4801 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
     4861 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
     4921 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
     4981 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
     5041 tctagggtta aggatccgca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5101 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5161 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5221 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5281 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5341 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5401 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5461 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5521 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5581 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5641 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5701 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     5761 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5821 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5881 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5941 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6001 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6061 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6121 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6181 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6241 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6301 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6361 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6421 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6481 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6541 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6601 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6661 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6721 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6781 aggggggcgg agcctatgga aaaacgccag caacgcgg
//
