LOCUS       sequence-275002-        8485 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty sgRNA expression vector for expression of sgRNA for Sp Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8485)
  TITLE     Kingsbury Lab Plasmids
REFERENCE   2  (bases 1 to 8485)
  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"
COMMENT     Sequence Label: pTJK459
FEATURES             Location/Qualifiers
     source          1..8485
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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)."
     primer_bind     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     134..154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        182..408
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             409..589
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    636..761
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1254..1487
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1672..1716
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        complement(2100..2348)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2158..2177)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2328..2348)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2379..2495
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2699..3881
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2930..3872
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3829..3849
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    4510..5098
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4563..4583)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4981..4992)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5185..5418
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5490..5611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5527..5546)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5581..5600
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5651..5786
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5806..5825)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5807..5825)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5835..5852)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5835..5851)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5844..5866)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5993..6448
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6080..6099)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6290..6311
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6474..6578
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6579..6647,6648..7439)
                     /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     complement(6797..6816)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7610..8198
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8099..8118
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8352..8369
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      181 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      241 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      301 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      361 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      421 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      481 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      541 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      601 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      661 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      721 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      781 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      841 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      901 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
      961 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1021 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1081 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1141 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1201 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1261 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1321 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1381 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1441 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1501 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1561 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1621 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1681 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1741 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1801 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1861 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     1921 ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg gtatcggtta
     1981 acactagaaa aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt
     2041 atttaaactt gctatgctgt ttccagcata gctcttaaac gagacgcatt aatcgtctcg
     2101 gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg
     2161 gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat
     2221 tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt
     2281 ctaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat
     2341 aggccctctt cctgcccgac cttggtaccg gatccaactt ttaaaagaaa aggggggatt
     2401 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     2461 gaattacaaa aacaaattac aaaattcaaa attttatcga tgcctccccg tcaccacccc
     2521 ccccaacccg ccccgaccgg agctgagagt aattcataca aaaggactcg cccctgcctt
     2581 ggggaatccc agggaccgtc gttaaactcc cactaacgta gaacccagag atcgctgcgt
     2641 tcccgccccc tcacccgccc gctctcgtca tcactgaggt ggagaagagc atgcgtgagg
     2701 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     2761 aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     2821 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     2881 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt
     2941 gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac
     3001 ttccacgccc ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
     3061 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
     3121 ggcttgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
     3181 ctttcgataa gtctctagcc atttaaaatt tttgatgata tcctgcgacg cttttttttt
     3241 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
     3301 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
     3361 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
     3421 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     3481 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     3541 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     3601 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     3661 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     3721 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     3781 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     3841 cagtggttca aagttttttt cttccatttc aggtgtcgtg aaaactaccc ctctagtacc
     3901 gccatgaccg agtacaagcc tactgttcgg ctcgcgactc gcgatgatgt ccccagagca
     3961 gtgagaacac tggctgctgc ctttgccgat taccccgcaa cccgccatac cgtggatccc
     4021 gaccggcaca tcgagagggt gactgaactg caggaattgt ttttgacaag ggtgggactc
     4081 gacattggaa aggtgtgggt ggccgatgac ggagccgccg tcgccgtttg gaccacacca
     4141 gagagcgtcg aagccggcgc ggtgttcgcc gagatcggcc caagaatggc cgagctctct
     4201 ggatctagac tggccgcgca acagcagatg gaaggcctgc tcgcgccaca ccgacctaag
     4261 gagcccgcat ggttcctggc caccgtaggc gtttcccccg atcaccaggg aaaggggctt
     4321 ggcagcgccg tagttctgcc cggcgtggag gccgctgaac gagctggtgt tcccgcattt
     4381 cttgagacca gtgcacctag gaaccttcct ttctatgaaa ggttgggttt caccgtcacc
     4441 gccgatgtgg aagtgcctga gggacctcga acctggtgca tgactcgaaa gcctggcgcc
     4501 taactcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4561 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     4621 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     4681 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     4741 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     4801 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     4861 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca
     4921 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     4981 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     5041 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct
     5101 ggaattcgag ctcggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     5161 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc
     5221 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     5281 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     5341 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     5401 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg
     5461 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     5521 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5581 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     5641 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     5701 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     5761 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     5821 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     5881 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     5941 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     6001 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     6061 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     6121 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     6181 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     6241 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     6301 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     6361 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     6421 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     6481 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6541 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6601 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6661 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6721 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6781 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6841 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6901 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6961 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     7021 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     7081 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     7141 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7201 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7261 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     7321 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7381 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7441 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7501 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7561 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7621 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7681 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7741 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7801 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7861 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7921 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7981 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     8041 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     8101 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     8161 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     8221 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     8281 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     8341 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     8401 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     8461 aagcgggcag tgagcgcaac gcaat
//
