LOCUS       sequence-352662-        8674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Neomycin (bU6), Ampk1 (mU6) and Ampk2(hU6) gRNAs and 
            Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8674)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8674)
  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: bU6-sgNeo1-mU6-sgAmpk1-hU6-sgAmpk2
FEATURES             Location/Qualifiers
     source          1..8674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             238..418
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(480..1068)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(560..579)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1239..2030,2031..2099))
                     /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     1862..1881
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2100..2204)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2279..2298)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2470..2849
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2851..3049
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3004..3024
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3067..3247
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3294..3419
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3916..4149
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4334..4378
                     /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_feature    4676..4793
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5530..5550
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5776..5796
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5947..5966
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6187..6686
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6222..6245)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6619..6638
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6690..6710
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6711..7739
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    7795..8383
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7848..7868)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8266..8277)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8386..8402)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8387..8403)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 cagaactaca caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag
       61 ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg
      121 ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg
      181 tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg
      241 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      301 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      361 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagc
      421 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      481 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      541 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      601 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      661 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      721 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
      781 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
      841 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
      901 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
      961 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1021 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1081 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1141 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     1201 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     1261 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     1321 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     1381 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     1441 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     1501 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     1561 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     1621 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     1681 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     1741 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     1801 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     1861 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     1921 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     1981 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     2041 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     2101 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     2161 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     2221 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     2281 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     2341 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     2401 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     2461 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     2521 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     2581 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     2641 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     2701 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     2761 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     2821 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     2881 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     2941 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     3001 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc
     3061 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     3121 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     3181 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     3241 tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc
     3301 gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga
     3361 gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca
     3421 gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa
     3481 agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag
     3541 ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac
     3601 catcccttca gacaggatca gaagaactta gatcattata taatacagta gcaaccctct
     3661 attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg
     3721 aagagcaaaa caaaagtaag accaccgcac agcaagcggc cggccgctga tcttcagacc
     3781 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     3841 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     3901 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     3961 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     4021 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     4081 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     4141 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     4201 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     4261 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     4321 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     4381 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     4441 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     4501 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc
     4561 cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa
     4621 cggatcggca ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta
     4681 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
     4741 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
     4801 attacaggga cagcagagat ccagtttggt tagtaccggg cccgctctag ccctgcagga
     4861 actgtttaaa ctgtcgtgac cgagcttgtc tgcccgcgca gtccactaga cagacgcgca
     4921 gaggtcgggc gggcctaggg ccaatttccc atggttccct tcatttgcat atatgatgta
     4981 ataaggttat ggagactatt aaacttagcc ctaatcaaac tatatgatga taatgtgtgt
     5041 ggtacaaaag gtcataactt attatatact ttgaaactta aaaaagggtt acagtttagt
     5101 caccataact gtaaaatttt ttctattctt agctttatat agttcttgag aggccatgtt
     5161 tatggtcatg gctgatgcaa tgcgggttta agagctaagc tggaaacagc atagcaagtt
     5221 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttcc
     5281 agtctgacta tcacgattga tccgacgcgc catctctagg cccgcgccgg ccccctcgca
     5341 cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat ataatatttc
     5401 ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct ttttaatact
     5461 agctacattt tacatgatag gcttggattt ctataagaga tacaaatact aaattattat
     5521 tttaaaaaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg tgttttgaga
     5581 ctataagtat cccttggaga accaccttgt tggcaaggct ccgaatcttc tgcgtttgag
     5641 agctaagcag aaagctgcat agcaagttca aataaggcta gtccgtacac aacttgaaaa
     5701 agtggcagcc gagtcggctg ctttttttgc tcgaatctac actcagctat ggcgcgcccc
     5761 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac
     5821 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     5881 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     5941 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     6001 atcttgtgga aagccagaaa catggccaat tgacaggcca taaaggtttc agagctaagc
     6061 acaagagtgc atagcaagtt gaaataaggc tagtccgttt acaacttgaa aaagtggcac
     6121 ccgagtcggg tgcttttttt gtttggcgcg cctatggctt aattaacatg gcggccgcat
     6181 tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
     6241 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
     6301 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc
     6361 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag
     6421 tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag
     6481 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
     6541 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
     6601 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
     6661 cctcttcctc atctccggga cccgggatgc ccaagaagaa gaggaaggtg tccaatttac
     6721 tgaccgtaca ccaaaatttg cctgcattac cggtcgatgc aacgagtgat gaggttcgca
     6781 agaacctgat ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc
     6841 ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc
     6901 ccgcagaacc tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag
     6961 taaaaactat ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tccgggctgc
     7021 cacgaccaag tgacagcaat gctgtttcac tggttatgcg gcggatccga aaagaaaacg
     7081 ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg
     7141 ttcgttcact catggaaaat agcgatcgct gccaggatat acgtaatctg gcatttctgg
     7201 ggattgctta taacaccctg ttacgtatag ccgaaattgc caggatcagg gttaaagata
     7261 tctcacgtac tgacggtggg agaatgttaa tccatattgg cagaacgaaa acgctggtta
     7321 gcaccgcagg tgtagagaag gcacttagcc tgggggtaac taaactggtc gagcgatgga
     7381 tttccgtctc tggtgtagct gatgatccga ataactacct gttttgccgg gtcagaaaaa
     7441 atggtgttgc cgcgccatct gccaccagcc agctatcaac tcgcgccctg gaagggattt
     7501 ttgaagcaac tcatcgattg atttacggcg ctaaggatga ctctggtcag agatacctgg
     7561 cctggtctgg acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt
     7621 caataccgga gatcatgcaa gctggtggct ggaccaatgt aaatattgtc atgaactata
     7681 tccgtaacct ggatagtgaa acaggggcaa tggtgcgcct gctggaagat ggcgattagc
     7741 catgtttaag ggttccggtt ccactaggta caattcgata tcaagcttat cgataatcaa
     7801 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     7861 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     7921 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     7981 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     8041 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     8101 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     8161 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     8221 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     8281 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     8341 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct
     8401 cgatcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc
     8461 tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca
     8521 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     8581 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg
     8641 gatctaccac acacaaggct acttccctga ttgg
//
