LOCUS       pgRNA_hMECP2_pro        8319 bp DNA     circular SYN 04-DEC-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8319)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 4, 2022 from SnapGene 6.1.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    15..317
                     /label=mU6 promoter (modified)
     protein_bind    209..242
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    257..273
                     /label=Sequencing Primer_SL-51
     misc_feature    318..329
                     /label=BstX I site
     misc_feature    350..442
                     /label=sgRNA scafold_93nt (F+E)
     misc_feature    443..448
                     /label=Xho I site
     enhancer        560..863
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        864..1067
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    1101..1697
                     /label=puro
     CDS             1101..1697
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    1236..1255
                     /label=qPCR For
     misc_feature    1421..1439
                     /label=qPCR rev
                     /label=qPCR rev(1)
     misc_feature    1704..1757
                     /label=t2A
     CDS             1707..1760
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      1767..1776
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1773..2483
                     /label=mCherry
     CDS             1773..2483
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     misc_feature    2301..2320
                     /label=qPCR for
     misc_feature    2380..2399
                     /label=qPCR rev
     protein_bind    2503..2536
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    2592..3180
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(3063..3074)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3183..3199)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             3709..3889
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3951..4539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4710..5570)
                     /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"
     promoter        complement(5571..5675)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5950..6329
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6331..6529
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6547..6727
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6774..6899
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7398..7631
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7816..7860
                     /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    8158..8275
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 gggcccgctc tagagatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacaga
       61 cttgtgggag aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag
      121 taactataga ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct
      181 acattttaca tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt
      241 ataaacagca caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat
      301 aagtatccct tggagaacca ccttgttggc agccaatcaa cagctggagg tttaagagct
      361 atgctggaaa cagcatagca agtttaaata aggctagtcc gttatcaact tgaaaaagtg
      421 gcaccgagtc ggtgcttttt ttctcgagta ctaggatcca ttaggcggcc gcgtggataa
      481 ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata
      541 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      601 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      661 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      721 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      781 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      841 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      901 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      961 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     1021 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
     1081 gtcagatccg ctagcccacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     1141 acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     1201 gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     1261 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     1321 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     1381 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     1441 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     1501 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     1561 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     1621 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     1681 cccgcaagcc cggtgccgga tcgggagagg gcagaggaag tctgctaaca tgcggtgacg
     1741 tcgaggagaa tcctggccca ccggtcgcca ccatggtgag caagggcgag gaggataaca
     1801 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     1861 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     1921 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     1981 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     2041 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     2101 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     2161 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     2221 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     2281 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     2341 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     2401 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     2461 gcatggacga gctgtacaag taagaattcg tcgagggacc taataacttc gtatagcata
     2521 cattatacga agttatacat gtttaagggt tccggttcca ctaggtacaa ttcgatatca
     2581 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2641 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2701 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     2761 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     2821 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     2881 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     2941 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     3001 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3061 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3121 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3181 atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat
     3241 acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt
     3301 tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     3361 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     3421 gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac
     3481 acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca
     3541 gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct
     3601 gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc
     3661 cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg
     3721 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     3781 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     3841 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca
     3901 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3961 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4021 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4081 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4141 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4201 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4261 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4321 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4381 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4441 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4501 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4561 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4621 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4681 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4741 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4801 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4861 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4921 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4981 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5041 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5101 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5161 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5221 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5281 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5341 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5401 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5461 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5521 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5581 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5641 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5701 gacgcttaag cggtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     5761 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     5821 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     5881 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     5941 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     6001 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     6061 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     6121 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     6181 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     6241 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     6301 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     6361 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     6421 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     6481 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc
     6541 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     6601 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     6661 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     6721 tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc
     6781 gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga
     6841 gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca
     6901 gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa
     6961 agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag
     7021 ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac
     7081 catcccttca gacaggatca gaagaactta gatcattata taatacagta gcaaccctct
     7141 attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg
     7201 aagagcaaaa caaaagtaag accaccgcac agcaagcggc cggccgcgct gatcttcaga
     7261 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     7321 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     7381 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     7441 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     7501 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     7561 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     7621 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     7681 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     7741 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     7801 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     7861 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     7921 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     7981 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg
     8041 cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg
     8101 aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt
     8161 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     8221 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     8281 ttattacagg gacagcagag atccagtttg gttagtacc
//
