LOCUS       sequence-177434-        5809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA expression vector for Evx1as RNA tethering assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5809)
  AUTHORS   Luo S, Lu JY, Liu L, Yin Y, Chen C, Han X, Wu B, Xu R, Liu W, Yan P,
            Shao W, Lu Z, Li H, Na J, Tang F, Wang J, Zhang YE, Shen X
  TITLE     Divergent lncRNAs Regulate Gene Expression and Lineage 
            Differentiation in Pluripotent Cells.
  JOURNAL   Cell Stem Cell. 2016 May 5;18(5):637-52. doi: 
            10.1016/j.stem.2016.01.024. Epub 2016 Mar 17.
  PUBMED    26996597
REFERENCE   2  (bases 1 to 5809)
  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: 
            "10.1016/j.stem.2016.01.024"; journalName: "Cell Stem Cell"; date: 
            "2016-05-5- 5"; volume: "18"; issue: "5"; pages: "637-52"
COMMENT     Sequence Label: CMV-sgE1TSS-short-3'box
FEATURES             Location/Qualifiers
     source          1..5809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_RNA        857..932
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      1679..2107
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1766..1785)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1976..1997
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2116..2136)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2121..2450
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2301..2436
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2363..2382
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2517..3311
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2571..2590)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3181..3200
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3485..3606
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3522..3541)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3576..3595
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3655..3671)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3655..3671)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3668..3690)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3679..3695
                     /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(3703..3709,3710..3727,3728..3733))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3748..3769
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3886..3903)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4057..4642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4137..4156)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4813..5604,5605..5673))
                     /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     5436..5455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5674..5778)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaagaaa
      841 ggagacccgc agctaagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
      901 atcaacttga aaaagtggca ccgagtcggt gcttttgaaa ggagacccgc agctaatggc
      961 tcggcagtga tgcgccaggt gtgggcctcg ctcgaatggg gaggagagtg tgaaaacaga
     1021 gagacacaca cactgggaaa gggagacacc cagacacaga ggatgcaaat ggaattccgc
     1081 acacagaaaa gggagcgctt aaggcgcgca gcgagttcca caggggcaac aggacttgat
     1141 ggcctgattt gaatttggat tggctcttca tagaagaaaa catcttttgg tttggcttat
     1201 ctccgggcta tttcaggtta tttactgagc agcggaagaa ggaaggaaat ttctaatagc
     1261 gcgtgaagca aggtaaccct tcgaaagcaa agagaggtga cagccacctt agtggagtcc
     1321 gccccggtgc atctctccgc tgcagccccg ccgacttgca gtccttctcc tgctggggcg
     1381 atcggctcag atgcggccag ttcttcgagg attggaattc tctgaacttc aaacgctgtg
     1441 ggtttggctt ccgcccggct aggagggata gagggtggct gagaatccta gggagccaag
     1501 gggagataag tcgcgccagt cgttttaaat ggaatctaaa cctttgcttg ggccaccact
     1561 ttctggagtt tcaaaagtag actgtacgct aagggtcata tctttttttg tttggtttgt
     1621 gtcttggttg gcgtcttaaa ggcggaaaga accagctggg gctctagggg gtatccccac
     1681 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1741 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1801 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1861 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1921 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1981 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2041 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2101 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2161 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2221 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2281 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2341 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     2401 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     2461 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     2521 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     2581 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     2641 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     2701 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     2761 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     2821 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     2881 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     2941 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3001 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     3061 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3121 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3181 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3241 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3301 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     3361 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3421 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3481 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     3541 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3601 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     3661 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3721 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3781 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3841 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3901 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3961 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4021 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4081 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4141 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4201 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4261 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4321 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4381 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4441 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4501 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4561 ttgatccggc aaacaaacca ccgctggtag cggttttttt gtttgcaagc agcagattac
     4621 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4681 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4741 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4801 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4861 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4921 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4981 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5041 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5101 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5161 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5221 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5281 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5341 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5401 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5461 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5521 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5581 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5641 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5701 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5761 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
