LOCUS       sequence-275062-        8476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SIX1 CRISPRi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8476)
  TITLE     Kingsbury Lab Plasmids
REFERENCE   2  (bases 1 to 8476)
  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: pTJK642
FEATURES             Location/Qualifiers
     source          1..8476
                     /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"
     misc_RNA        complement(1995..2070)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2091..2339)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2149..2168)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2319..2339)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2370..2486
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2690..3872
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2921..3863
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3820..3840
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    4501..5089
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4554..4574)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4972..4983)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5176..5409
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5481..5602
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5518..5537)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5572..5591
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5642..5777
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5797..5816)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5798..5816)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5826..5843)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5826..5842)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5835..5857)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5984..6439
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6071..6090)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6281..6302
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6465..6569
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6570..6638,6639..7430)
                     /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(6788..6807)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7601..8189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8090..8109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8343..8360
                     /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 attttaactt gctatttcta gctctaaaac cggcaactag cagcatccac ggtgtttcgt
     2101 cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac ggtaagcata
     2161 tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa ttattacttt
     2221 ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat tctaattatc
     2281 tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa taggccctct
     2341 tcctgcccga ccttggtacc ggatccaact tttaaaagaa aaggggggat tggggggtac
     2401 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     2461 aaacaaatta caaaattcaa aattttatcg atgcctcccc gtcaccaccc cccccaaccc
     2521 gccccgaccg gagctgagag taattcatac aaaaggactc gcccctgcct tggggaatcc
     2581 cagggaccgt cgttaaactc ccactaacgt agaacccaga gatcgctgcg ttcccgcccc
     2641 ctcacccgcc cgctctcgtc atcactgagg tggagaagag catgcgtgag gctccggtgc
     2701 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     2761 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     2821 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     2881 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
     2941 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacgcc
     3001 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt
     3061 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcttgggc
     3121 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata
     3181 agtctctagc catttaaaat ttttgatgat atcctgcgac gctttttttt tggcaagata
     3241 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg
     3301 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc
     3361 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg
     3421 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt
     3481 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc
     3541 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag
     3601 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct
     3661 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc
     3721 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct
     3781 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc
     3841 aaagtttttt tcttccattt caggtgtcgt gaaaactacc cctctagtac cgccatgacc
     3901 gagtacaagc ctactgttcg gctcgcgact cgcgatgatg tccccagagc agtgagaaca
     3961 ctggctgctg cctttgccga ttaccccgca acccgccata ccgtggatcc cgaccggcac
     4021 atcgagaggg tgactgaact gcaggaattg tttttgacaa gggtgggact cgacattgga
     4081 aaggtgtggg tggccgatga cggagccgcc gtcgccgttt ggaccacacc agagagcgtc
     4141 gaagccggcg cggtgttcgc cgagatcggc ccaagaatgg ccgagctctc tggatctaga
     4201 ctggccgcgc aacagcagat ggaaggcctg ctcgcgccac accgacctaa ggagcccgca
     4261 tggttcctgg ccaccgtagg cgtttccccc gatcaccagg gaaaggggct tggcagcgcc
     4321 gtagttctgc ccggcgtgga ggccgctgaa cgagctggtg ttcccgcatt tcttgagacc
     4381 agtgcaccta ggaaccttcc tttctatgaa aggttgggtt tcaccgtcac cgccgatgtg
     4441 gaagtgcctg agggacctcg aacctggtgc atgactcgaa agcctggcgc ctaactcgac
     4501 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     4561 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     4621 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     4681 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     4741 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     4801 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     4861 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
     4921 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     4981 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     5041 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggaattcga
     5101 gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     5161 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     5221 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     5281 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     5341 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     5401 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     5461 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     5521 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5581 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     5641 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5701 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5761 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     5821 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     5881 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     5941 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     6001 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     6061 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     6121 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     6181 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     6241 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     6301 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     6361 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     6421 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     6481 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6541 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     6601 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6661 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6721 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6781 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6841 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6901 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6961 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     7021 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     7081 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     7141 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     7201 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     7261 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     7321 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     7381 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     7441 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     7501 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7561 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     7621 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     7681 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7741 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7801 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     7861 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     7921 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7981 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     8041 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     8101 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     8161 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     8221 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     8281 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     8341 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     8401 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     8461 gtgagcgcaa cgcaat
//
