LOCUS       sequence-487657-        7026 bp    DNA     circular SYN 01-OCT-2025
DEFINITION  Express Eco3RT from a CAG promoter; also express an msr and HEK3, 
            FANCF and EMX1 retron msd donor-gRNAs from a single H1 promoter, 
            separated by tRNA-Cys-GCA sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7026)
  AUTHORS   Gonzalez-Delgado A, Lopez SC, Rojas-Montero M, Fishman CB, Shipman 
            SL
  TITLE     Simultaneous multi-site editing of individual genomes using retron 
            arrays.
  JOURNAL   bioRxiv [Preprint]. 2023 Jul 17:2023.07.17.549397. doi: 
            10.1101/2023.07.17.549397.
  PUBMED    37503029
REFERENCE   2  (bases 1 to 7026)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2023 Jul 17:2023.07.17.549397. doi: 
            10.1101/2023.07.17.549397."
COMMENT     Sequence Label: pSCL758
FEATURES             Location/Qualifiers
     source          1..7026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        368..646
                     /label=chicken beta-actin promoter
     intron          647..1663
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1587..1609
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1671..1690
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2691..2711
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2752..2771)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2796..2844
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        3044..3258
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3179..3198
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        3652..3727
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        4024..4099
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(4511..4527)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4511..4527)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4524..4546)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4535..4551
                     /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(4559..4565,4566..4583,4584..4589))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4604..4625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4684..4879
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4730..4865
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4885..5019
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4922..4941)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4976..4995
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5087..5104)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5258..5846)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5338..5357)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6017..6808,6809..6877))
                     /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     6640..6659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6878..6982)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(7013..7026,1..366)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc ccccctcccc
      421 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      481 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
      541 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      601 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
      661 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
      721 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
      781 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
      841 ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
      901 ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc
      961 ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg
     1021 gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg
     1081 agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt
     1141 tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc
     1201 cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc
     1261 cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc
     1321 ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt
     1381 gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg
     1441 cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc
     1501 gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg
     1561 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
     1621 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
     1681 ggttattgtg ctgtctcatc attttggcaa agaattgagc tcgatatcgg tacgccacca
     1741 tgcgcattta ctctctgatc gacagccaaa ccttaatgac caaagggttc gcatccgagg
     1801 tcatgaggag cccagaaccc cctaagaagt gggacattgc gaagaagaag ggcggaatgc
     1861 gtacgatata ccatccctct tctaaggtga agctgataca gtactggctg atgaacaacg
     1921 tgttctccaa attgccgatg cacaacgccg cgtacgcttt cgtgaagaat agatctatca
     1981 agtctaacgc actgctgcac gcagagagta agaacaaata ctacgttaag attgacctga
     2041 aggacttctt tccaagcatc aagttcacag acttcgaata tgcctttacc cggtaccgtg
     2101 acagaataga gttcacgacc gagtacgaca aagaactgct tcagctgatt aagaccattt
     2161 gtttcatttc tgactctaca ctgccaatag gcttccccac ttcccctctt atagccaatt
     2221 tcgtcgccag ggagctggac gagaagctca ctcagaagct gaacgctata gacaagctca
     2281 acgctacgta cactcgctac gcagacgaca taatcgtgag cacgaacatg aagggcgcct
     2341 ctaagctgat cttagactgc ttcaagcgga ccatgaagga aatcggaccc gatttcaaga
     2401 tcaatatcaa gaagttcaaa atatgctctg ccagtggcgg ctcaattgtc gtgacgggcc
     2461 ttaaggtctg tcatgacttc cacataactc tgcaccggtc tatgaaggac aagatccgcc
     2521 tgcacctctc tctcctgtcc aaaggtattc tgaaggacga ggaccacaac aagctgtccg
     2581 ggtacatcgc ctacgctaag gacatcgatc cacacttcta caccaagctc aataggaagt
     2641 acttccagga gatcaagtgg atacaaaacc tgcataataa ggtggagcca ccaaagaaga
     2701 aaagaaaggt ctgatcccgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg
     2761 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     2821 gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     2881 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     2941 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     3001 ctgccatagc agatctgcga aatcggatcc agcggccaat tcggaacgct gacgtcatca
     3061 acccgctcca aggaatcgcg ggcccagtgt cactaggcgg gaacacccag cgcgcgtgcg
     3121 ccctggcagg aagatggctg tgagggacag gggagtggcg ccctgcaata tttgcatgtc
     3181 gctatgtgtt ctgggaaatc accataaacg tgaaatgtct ttggatttgg gaatcttata
     3241 agttctgtat gagaccactg ataagattcc gtaagagcca aacctagcat tttatgggtt
     3301 aatagcccat cgggccatga gtcatggttt cgcctagtat tttagctatt tttttagagg
     3361 gggtatagct cagtggtaga gcatttgact gcagatcaag aggtccccgg ttcaaatccg
     3421 ggtgccccct agctatgccc gtcgttcagt tcgctgactt ctccagccct ggcctgggtc
     3481 aatccttggg gcccagactg agcacgtgct aacagaggaa aggaagccct gcttcctcca
     3541 gagggcgtcg caggacagct tttcctagac aggggcttca gcgaactgat cgacgtgctc
     3601 aagtaggttt ggctcttacg gaatcttatc aggcccagac tgagcacgtg agttttagag
     3661 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     3721 tcggtgcaga gggggtatag ctcagtggta gagcatttga ctgcagatca agaggtcccc
     3781 ggttcaaatc cgggtgcccc ctagctatgc ccgtcgttca gttcgctgag aaagcggaag
     3841 tagggccttc gcgcacctca tggaatccct tctgcagcat ctagatcgct tttccgagct
     3901 tctggcggtc tcaagcacta cctacgtcag cacctgggac cccgccacct cagcgaactg
     3961 atcgacgtgc tcaagtaggt ttggctctta cggaatctta tcaggaatcc cttctgcagc
     4021 accgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     4081 agtggcaccg agtcggtgca gagggggtat agctcagtgg tagagcattt gactgcagat
     4141 caagaggtcc ccggttcaaa tccgggtgcc ccctagctat gcccgtcgtt cagttcgctg
     4201 aacaaacggc agaagctgga ggaggaaggg cctgagtccg agcagaagaa aaagttctcc
     4261 catcacatca accggtggcg cattgccacg aagcaggcca atggggagga catcgatgtc
     4321 atcagcgaac tgatcgacgt gctcaagtag gtttggctct tacggaatct tatcagagtc
     4381 cgagcagaag aagaagtttc agagctatgc tggaaacagc atagcaagtt gaaataaggc
     4441 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttt tttagcttgg
     4501 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4561 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4621 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga
     4681 tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4741 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4801 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4861 aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa
     4921 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4981 ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc
     5041 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5101 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5161 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5221 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5281 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5341 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5401 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5461 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5521 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5581 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5641 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5701 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5761 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5821 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5881 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5941 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6001 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6061 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6121 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6181 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6241 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6301 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6361 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6421 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6481 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6541 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6601 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6661 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6721 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6781 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6841 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6901 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6961 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctggg tcgacattga
     7021 ttattg
//
