LOCUS       sequence-375209-        4973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA sequence provider (for checkpoint library).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4973)
  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 4973)
  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: Pro-tRNA-gRNA scaffold for library 2
FEATURES             Location/Qualifiers
     source          1..4973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..441
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        442..645
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     596..616
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      990..999
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(996..998,999..1001,1002..1715)
                     /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(996..998,999..1001,1002..1712)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1041..1062)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1302..1321)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1649..1670
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1838..1959
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1875..1894)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1929..1948
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1966..2421)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2103..2124)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2315..2334
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2448..2552
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2554..2911
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2554..2745
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2577..2597)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2762..2897
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2946..3740
                     /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             2946..3740
                     /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(3000..3019)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3610..3629
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3928..3947)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3972..4019
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4348..4936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4837..4856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
       61 gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc
      121 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      181 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      241 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      301 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      361 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      421 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      481 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      541 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      601 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
      661 cagatccgct agcgctaccg gactcagatc tcgagctcgg agcatgtctt ctcgctcttc
      721 ggtttcagag ctatgccggc gagacggcgc cgcgcgtctc taaccggcat agcaagttga
      781 aataaggcta gtccgttatc aacttggccc gcgaagcagg tgatgcgcat atgcgttagc
      841 ttcacctgca aatcgggcca agtggcaccg agtcggtgct ggctcgttgg tctaggggta
      901 tgattctcgc ttagggtgcg agaggtcccg ggttcaaatc ccggacgagc cccgaagagc
      961 gcgggaagac tcgagcggat ccaccggtcg ccaccatggt gagcaagggc gaggagctgt
     1021 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1081 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1141 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1201 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1261 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1321 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1381 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1441 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1501 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1561 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     1621 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1681 ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat
     1741 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     1801 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     1861 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     1921 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     1981 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     2041 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     2101 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     2161 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     2221 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     2281 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     2341 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     2401 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2461 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2521 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     2581 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2641 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     2701 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     2761 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2821 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     2881 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     2941 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3001 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3061 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3121 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3181 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3241 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3301 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     3361 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     3421 acgaggagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     3481 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     3541 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     3601 aggacatagc gttggctacc cgtgatattg ctgaggagct tggcggcgaa tgggctgacc
     3661 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     3721 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     3781 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     3841 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     3901 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     3961 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     4021 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gagcccattg
     4081 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     4141 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     4201 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4261 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4321 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4381 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4441 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4501 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4561 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4621 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4681 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4741 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4801 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4861 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4921 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg cct
//
