LOCUS       sequence-375211-        4898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA sequence provider (for checkpoint and immune library).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4898)
  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 4898)
  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: Gln-tRNA-gRNA scaffold for library 1&2
FEATURES             Location/Qualifiers
     source          1..4898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        104..407
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        408..611
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     562..582
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      881..890
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(887..889,890..892,893..1606)
                     /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(887..889,890..892,893..1603)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(932..953)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1193..1212)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1540..1561
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1729..1850
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1766..1785)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1820..1839
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1857..2312)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1994..2015)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2206..2225
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2339..2443
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2445..2802
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2445..2636
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2468..2488)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2653..2788
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2837..3631
                     /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             2837..3631
                     /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(2891..2910)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3501..3520
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3819..3838)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3863..3910
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4239..4827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4728..4747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
       61 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      121 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      181 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      241 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      301 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      361 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      421 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      481 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      541 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      601 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact cagatctcga
      661 gctcggagca tcgagacggg tctcggttta agagctatgc tggaaacagc atagcaagtt
      721 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctggttccat
      781 ggtgtaatgg ttagcactct ggactctgaa tccagcgatc cgagttcaaa tctcggtgga
      841 acctcgagac cgcgcgtctc atcgagcgga tccaccggtc gccaccatgg tgagcaaggg
      901 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      961 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1021 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1081 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1141 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1201 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1261 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1321 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1381 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1441 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1501 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1561 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg gccgcgactc
     1621 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     1681 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     1741 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     1801 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat
     1861 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     1921 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     1981 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2041 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2101 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2161 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2221 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2281 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     2341 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2401 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     2461 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     2521 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     2581 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     2641 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     2701 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     2761 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     2821 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2881 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     2941 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3001 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3061 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3121 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3181 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3241 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3301 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3361 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3421 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3481 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3541 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     3601 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     3661 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3721 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3781 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     3841 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     3901 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     3961 agagcccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     4021 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     4081 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4141 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4201 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4261 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4321 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4381 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4441 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4501 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4561 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4621 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4681 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4741 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4801 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4861 gccttttgct ggccttttgc tcacatgttc tttcctgc
//
