LOCUS       sequence-341739-        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   25..87
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        390..1568
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          621..1559
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1516..1536
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     ncRNA           1585..1621
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        1642..1717
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           1969..2036
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(2046..2063)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2052..2259
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        2326..2629
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2630..2833
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2784..2804
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2856..2865
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2862..2864,2865..2867,2868..3578)
                     /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(2907..2928)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3168..3187)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3515..3536
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3579..3635
                     /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      3642..3651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3648..4358
                     /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(3793..3811)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    4375..4599
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4956..4990
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        5021..5125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5126..5194,5195..5986)
                     /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(5344..5363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6157..6745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6646..6665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatcccctga ttctctcgag ttaaccctag aaagataatc atattgtgac gtacgttaaa
       61 gataatcatg cgtaaaattg acgcatgtgt tttatcgatc tgtatatcga ggtttattta
      121 ttaatttgaa tagatattaa gttttattat atttacactt acatactaat aataaattca
      181 acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt
      241 ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact tattttgtac
      301 tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat acataataga
      361 aacaaattat acttattaat taatctagag gctccggtgc ccgtcagtgg gcagagcgca
      421 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
      481 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
      541 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
      601 ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta
      661 cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg
      721 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc
      781 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
      841 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt
      901 gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc
      961 tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc
     1021 gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
     1081 ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct
     1141 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
     1201 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt
     1261 cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg
     1321 agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt
     1381 taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg
     1441 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg
     1501 gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
     1561 tgtcgtgaac gcgtgctcgg actcctgatg agtccgtgag gacgaaacga gtaagctcgt
     1621 cgagtccgag cagaagaaga agttttagag ctagaaatag caagttaaaa taaggctagt
     1681 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcgca gagccaaacc tagcatttta
     1741 tgggttaata gcccatcggg ccatgagtca tggtttcgcc tagtatttta gctatgcccg
     1801 tcgttactag tacaaacggc agaagctgga ggaggaaggg cctgagtccg agcagaagaa
     1861 gcttaagggc tcccatcaca tcaaccggtg gcgcattgcc acgaagcagg ccaatgggga
     1921 ggacatcgat gtcacctagg atcgacgtgc tcaagtaggt ttggctctgg ccggcatggt
     1981 cccagcctcc tcgctggcgc cggctgggca acatgcttcg gcatggcgaa tgggacacgc
     2041 gtcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2101 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2161 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2221 gggggaggat tgggaagaga atagcaggca tgctggggaa agctttagtt attaatagta
     2281 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     2341 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     2401 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     2461 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     2521 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     2581 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     2641 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     2701 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     2761 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     2821 tataagcaga gctggtttag tgaaccgtca gatccgccac catggtgagc aagggcgagg
     2881 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2941 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3001 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3061 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3121 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3181 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3241 agggcatcga cttcaaggag gacggcaaca tttcagggca caagctggag tacaactaca
     3301 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3361 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     3421 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     3481 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     3541 ccgccgggat cactctcggc atggacgagc tgtacaaggc tactaacttc agcctgctga
     3601 agcaggctgg agacgtggag gagaaccctg gacctgctag cgccaccatg gtgagcaagg
     3661 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     3721 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     3781 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     3841 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     3901 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     3961 atgacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     4021 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaaa
     4081 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     4141 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     4201 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     4261 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     4321 gccactccac cggcggcatg gacgagctgt acaagtaact cttggcccgg atccctgtgc
     4381 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     4441 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     4501 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaaa
     4561 acaatagcag gcatgctggg gatgcggtgg gctctatggg tcgacggcgc gccctagacg
     4621 aagcagctcc agcctacaca atcgctcaag acgtgtaatg cttttattat atattagtca
     4681 cgatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat
     4741 aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc
     4801 attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca
     4861 cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc
     4921 gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat
     4981 ctttctaggg ttaaggatcc gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5041 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5101 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5161 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5221 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5281 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5341 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5401 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5461 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5521 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5581 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5641 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5701 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5761 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5821 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5881 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5941 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6001 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6061 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6121 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6181 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6241 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6301 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6361 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6421 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6481 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6541 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6601 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6661 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6721 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6781 cttttgctgg ccttttgctc acatgttctt tcctgcgt
//
