LOCUS       sequence-169088-        7858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7858)
  TITLE     pLKO variants
REFERENCE   2  (bases 1 to 7858)
  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: pLKO.3 Thy1.1
FEATURES             Location/Qualifiers
     source          1..7858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    55..76
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(91..96,97..114,115..121)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    129..145
                     /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     134..156
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     153..169
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     153..169
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     188..208
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        190..208
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        236..462
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             463..643
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    690..815
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1308..1541
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1726..1770
                     /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        2287..2535
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2287..2307
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2458..2477
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2627..2744
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2793..3303
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3222..3240
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3312..3800
                     /codon_start=1
                     /product="mouse GPI-anchored cell surface glycoprotein of 
                     the immunoglobulin superfamily"
                     /label=Thy1.1
                     /note="This allele of Thy1 is also known as CD90.1."
                     /translation="MNPAISVALLLSVLQVSRGQKVTSLTACLVNQNLRLDCRHENNTK
                     DNSIQHEFSLTREKRKHVLSGTLGIPEHTYRSRVTLSNQPYIKVLTLANFTTKDEGDYF
                     CELRVSGANPMSSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL"
     misc_feature    3819..4407
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3872..3892)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4290..4301)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4410..4426)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4411..4427)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4612..4845
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4917..5038
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4954..4973)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5008..5027
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5078..5213
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5233..5252)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5234..5252)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5262..5279)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5262..5278)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5271..5293)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5420..5875
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5507..5526)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5717..5738
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5901..6005
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6006..6074,6075..6866)
                     /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(6224..6243)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7037..7625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7526..7545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7779..7796
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
       61 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      121 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      181 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag
      241 tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta
      301 caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc
      361 ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat
      421 tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac
      481 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
      541 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
      601 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
      661 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
      721 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
      781 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
      841 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
      901 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
      961 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     1021 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     1081 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     1141 ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1201 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1261 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1321 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1381 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1441 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1501 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1561 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     1621 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     1681 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     1741 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     1801 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     1861 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     1921 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     1981 agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg atcacgagac
     2041 tagcctcgac cacgacgcaa tggagaaaga cggagagcgc caacggcgtc catctcgaag
     2101 gagtcgccag cgataaccgg agtagttgaa atggtaataa gacgaccaat ctgaccagca
     2161 aggaagccaa gatgggaaag gtcatgcggc atacgctcgg cgccagtttg aatattagac
     2221 ataatttatc ctcaagtaag ggggtacacg cgtcagtcga gtcctgtcga caaggtcggg
     2281 caggaagagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     2341 agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     2401 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     2461 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     2521 aaaggacgaa acaccggtcc gcaggtatgc ttgcgtgaat tccacctcga gctacttaat
     2581 taaaccagtt ttcgacctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     2641 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2701 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2761 acagcagaga tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc
     2821 agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc
     2881 gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc
     2941 gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc
     3001 ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc
     3061 gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat
     3121 agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg
     3181 gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag
     3241 cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc
     3301 cagggggatc catgaaccca gccatcagcg tcgctctcct gctctcagtc ttgcaggtgt
     3361 cccgagggca gaaggtgacc agcctgacag cctgcctggt gaaccaaaac cttcgcctgg
     3421 actgccgcca tgagaataac accaaggata actccatcca gcatgagttc agcctgaccc
     3481 gagagaagag gaagcacgtg ctctcaggca cccttgggat acccgagcac acgtaccgct
     3541 cccgcgtcac cctctccaac cagccctata tcaaggtcct taccctagcc aacttcacca
     3601 ccaaggatga gggcgactac ttttgtgagc ttcgcgtctc gggcgcgaat cccatgagct
     3661 ccaataaaag tatcagtgtg tatagagaca agctggtcaa gtgtggcggc ataagcctgc
     3721 tggttcagaa cacatcctgg atgctgctgc tgctgctttc cctctccctc ctccaagccc
     3781 tggacttcat ttctctgtga gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     3841 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3901 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3961 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     4021 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     4081 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     4141 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     4201 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     4261 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     4321 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     4381 ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgagac ctagaaaaac
     4441 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag
     4501 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga
     4561 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     4621 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     4681 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     4741 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     4801 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc
     4861 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
     4921 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4981 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5041 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     5101 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     5161 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
     5221 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     5281 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     5341 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     5401 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     5461 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     5521 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5581 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     5641 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5701 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     5761 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     5821 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     5881 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5941 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     6001 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     6061 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     6121 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     6181 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     6241 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6301 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6361 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6421 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6481 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6541 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6601 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6661 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6721 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6781 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6841 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6901 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6961 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     7021 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     7081 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     7141 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     7201 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     7261 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     7321 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     7381 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7441 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7501 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7561 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7621 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7681 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     7741 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     7801 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatg
//
