LOCUS       sequence-376682-        7820 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express -Eco1 AAVS1 editing ncRNA and gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7820)
  AUTHORS   Lopez SC, Crawford KD, Lear SK, Bhattarai-Kline S, Shipman SL
  TITLE     Precise genome editing across kingdoms of life using retron-derived 
            DNA.
  JOURNAL   Nat Chem Biol. 2022 Feb;18(2):199-206. doi: 
            10.1038/s41589-021-00927-y. Epub 2021 Dec 23.
  PUBMED    34949838
REFERENCE   2  (bases 1 to 7820)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41589-021-00927-y"; journalName: "Nat Chem Biol"; date: 
            "2022-02"; volume: "18"; issue: "2"; pages: "199-206"
COMMENT     Sequence Label: pSCL.180
FEATURES             Location/Qualifiers
     source          1..7820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2611..2825
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     2746..2765
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     LTR             3625..3805
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3831..3848)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3837..4061
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4107..4535
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4194..4213)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4404..4425
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4544..4564)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4549..4878
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4729..4864
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4791..4810
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4926..4973
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4992..5366
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    5496..5617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5533..5552)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5587..5606
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5666..5682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5666..5682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5679..5701)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5690..5706
                     /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(5714..5720,5721..5738,5739..5744))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5759..5780
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5897..5914)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6068..6656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6148..6167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6827..7618,7619..7687))
                     /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     7450..7469
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7688..7792)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatcg tacgaattcg gaacgctgac gtcatcaacc cgctccaagg
     2641 aatcgcgggc ccagtgtcac taggcgggaa cacccagcgc gcgtgcgccc tggcaggaag
     2701 atggctgtga gggacagggg agtggcgccc tgcaatattt gcatgtcgct atgtgttctg
     2761 ggaaatcacc ataaacgtga aatgtctttg gatttgggaa tcttataagt tctgtatgag
     2821 accacccgaa acgagctagc tcgtcatgat aagattccgt atgcgcaccc ttagcgagag
     2881 gtttatcatt aaggtcaacc tctggatgtt gtttcggcat cctgcattga atctgagtta
     2941 ctgtctgttt tccttaatgt ggctctggtt ctgggtactt ttatctgtcc cctccacccc
     3001 acaatggaac cactagggac aggattggtg acagaaaagc ccccatcctt aggcctcctc
     3061 cttcctagtc tcctaggaaa cccgtttctt ctgacgtaag ggtgcgcata cggaatctta
     3121 tcagtcccct ccaccccaca gtggtttcag agctatgctg gaaacagcat agcaagttga
     3181 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttgtacct
     3241 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     3301 ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac
     3361 cacacacaag gctacttccc tgattggcag aactacacac cagggccagg gatcagatat
     3421 ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa ggtagaagaa
     3481 gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg gatggatgac
     3541 ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca tcacatggcc
     3601 cgagagctgc atccggactg tactgggtct ctctggttag accagatctg agcctgggag
     3661 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     3721 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     3781 tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag cctcgactgt
     3841 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3901 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3961 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     4021 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     4081 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     4141 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     4201 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4261 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4321 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4381 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4441 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4501 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     4561 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4621 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4681 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4741 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4801 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4861 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag
     4921 cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
     4981 ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
     5041 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
     5101 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
     5161 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
     5221 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
     5281 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
     5341 acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct
     5401 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5461 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5521 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5581 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5641 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5701 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5761 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5821 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5881 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5941 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6001 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6061 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6121 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6181 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6241 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6301 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6361 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6421 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6481 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6541 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6601 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6661 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6721 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6781 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6841 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6901 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6961 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7021 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7081 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7141 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7201 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7261 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7321 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7381 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7441 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7501 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7561 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7621 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7681 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7741 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7801 cccgaaaagt gccacctgac
//
