LOCUS       sequence-375212-        4895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA sequence provider (for immune library).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4895)
  AUTHORS   Lu Z, Ni K, Wang Y, Zhou Y, Li Y, Yan J, Song Q, Liu M, Xu Y, Yu Z, 
            Guo T, Ma L
  TITLE     An in-library ligation strategy and its application in CRISPR/Cas9 
            screening of high-order gRNA combinations.
  JOURNAL   Nucleic Acids Res. 2022 Jun 24;50(11):6575-6586. doi: 
            10.1093/nar/gkac458.
  PUBMED    35670669
REFERENCE   2  (bases 1 to 4895)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2022-06-24- 24"; volume: "50"; issue: 
            "11"; pages: "6575-6586"
COMMENT     Sequence Label: Gly-tRNA-gRNA scaffold for library 1
FEATURES             Location/Qualifiers
     source          1..4895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        105..408
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        409..612
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     563..583
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_RNA        691..766
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      879..888
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(885..887,888..890,891..1604)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(885..887,888..890,891..1601)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(930..951)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1191..1210)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1538..1559
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1727..1848
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1764..1783)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1818..1837
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1855..2310)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1992..2013)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2204..2223
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2337..2441
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2443..2800
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2443..2634
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2466..2486)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2651..2786
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2835..3629
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2835..3629
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2889..2908)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3499..3518
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3817..3836)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3861..3908
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4237..4825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4726..4745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa
       61 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      121 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      181 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      241 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      301 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      361 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      421 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      481 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      541 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      601 ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggac tcagatctcg
      661 agctcggagc atcgagacgc acctgcgaag gttttagagc tagaaatagc aagttaaaat
      721 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctgca tgggtggttc
      781 agtggtagaa ttctcgcctg ccacgcggga ggcccgggtt cgattcccgg cccatgcaaa
      841 cggcaggtgg cgcgtctcat cgagcggatc caccggtcgc caccatggtg agcaagggcg
      901 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
      961 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1021 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1081 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1141 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1201 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1261 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1321 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1381 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1441 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     1501 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1561 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc cgcgactcta
     1621 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     1681 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     1741 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     1801 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg
     1861 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     1921 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     1981 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2041 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2101 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2161 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2221 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2281 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     2341 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2401 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     2461 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2521 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2581 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2641 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2701 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     2761 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     2821 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     2881 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     2941 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3001 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3061 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3121 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3181 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3241 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3301 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3361 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3421 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3481 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3541 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3601 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     3661 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     3721 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     3781 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     3841 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     3901 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     3961 agcccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     4021 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     4081 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4141 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4201 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4261 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4321 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4381 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4441 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4501 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4561 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4621 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4681 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4741 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4801 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4861 cttttgctgg ccttttgctc acatgttctt tcctg
//
