LOCUS       Exported                8451 bp ds-DNA     circular SYN 20-JAN-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Feb 7, 2025 from SnapGene Viewer 5.2.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8451
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      1..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        738..842
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             843..1703
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1:  843 ..  911 / #ccffcc / signal sequence
                        2:  912 .. 1703 / #ccffcc
                     Cleavage site after base 911"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1874..2462
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     promoter        2524..2764
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    2773..2873
                     /label=sgRNA_S1b
                     /note="color: #33cccc"
     enhancer        3572..3857
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
                     /note="color: #ffffff"
     promoter        3859..4136
                     /label=chicken beta-actin promoter
                     /note="color: #ffffff; direction: RIGHT"
     intron          4137..4364
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
                     /note="color: #------"
     CDS             4382..4438
                     /codon_start=1
                     /label=bpNLS
                     /note="color: #993366"
                     /translation="MKRTADGSEFESPKKKRKV"
     CDS             4439..5080
                     /codon_start=1
                     /label=DNMT3L
                     /note="color: #993366"
                     /translation="GPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGG
                     TLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQE
                     SQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKH
                     APLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL"
     CDS             5321..6904
                     /codon_start=1
                     /label=Dead eCas12f1
                     /note="color: #993366"
                     /translation="AKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKV
                     KEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQL
                     QKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSRVCYRRAAELFKNAAIASGLRS
                     KIKSNFRLKELKNMKSRLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRW
                     QVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGD
                     YQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIAVGVRSPLVCAINNAF
                     SRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLI
                     ERWACEIADFFIKNKVGTVQMENLEGMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQY
                     GIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENAAYNAALNIS
                     NPKLKSTKEEP"
     CDS             6950..7135
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /note="color: #993366"
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             7148..7195
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /note="color: #cc99b2"
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             7205..7261
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /note="color: #cc99b2
                     Cleavage site after base 7258"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7262..7981
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="This feature has 3 segments:
                        1: 7262 .. 7264 / #05fd14
                        2: 7265 .. 7267 / #05fd14 / 1a
                        3: 7268 .. 7981 / #05fd14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    8021..8228
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     repeat_region   8237..8377
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
ORIGIN
        1 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
      361 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
      481 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
      541 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
      601 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
      661 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
      721 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      781 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
      841 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
      901 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
      961 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     1021 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1081 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1141 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1201 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1261 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1321 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     1381 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     1441 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     1501 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     1561 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     1621 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     1681 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     1741 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     1801 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1861 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1921 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1981 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     2041 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2101 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2161 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2221 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2281 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2341 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2401 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2461 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2521 tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     2581 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     2641 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     2701 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     2761 ggacgaaaca ccgaccgctg gacttcgagt ccaggtgggc tgcttgcatc agcctaatgt
     2821 cgagaagtgc tttcttcgga aagtaaccct cgaaacaaag aaaggaatgc aacggagacc
     2881 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     2941 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     3001 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3061 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     3121 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     3181 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
     3241 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     3301 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     3361 acggccagtg aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc
     3421 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3481 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatagatccg
     3541 gtctcctttt attttttgtt ttagaggtac ccgttacata acttacggta aatggcccgc
     3601 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt
     3661 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     3721 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     3781 attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     3841 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
     3901 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
     3961 gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg
     4021 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
     4081 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
     4141 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
     4201 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
     4261 gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa
     4321 tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac
     4381 catgaaacgg acagccgacg gaagcgagtt cgagtcacca aagaagaagc ggaaagtcgg
     4441 ccctatggag atatacaaga cagtgtctgc atggaagaga cagccagtgc gggtactgag
     4501 cctcttcaga aacatcgaca aggtactaaa gagtttgggc ttcttggaaa gcggttctgg
     4561 ttctggggga ggaacgctga agtacgtgga agatgtcaca aatgtcgtga ggagagacgt
     4621 ggagaaatgg ggcccctttg acctggtgta cggctcgacg cagcccctag gcagctcttg
     4681 tgatcgctgt cccggctggt acatgttcca gttccaccgg atcctgcagt atgcgctgcc
     4741 tcgccaggag agtcagcggc ccttcttctg gatattcatg gacaatctgc tgctgactga
     4801 ggatgaccaa gagacaacta cccgcttcct tcagacagag gctgtgaccc tccaggatgt
     4861 ccgtggcaga gactaccaga atgctatgcg ggtgtggagc aacattccag ggctgaagag
     4921 caagcatgcg cccctgaccc caaaggaaga agagtatctg caagcccaag tcagaagcag
     4981 gagcaagctg gacgccccga aagttgacct cctggtgaag aactgccttc tcccgctgag
     5041 agagtacttc aagtattttt ctcaaaactc acttcctctt ggagggccga gctctggcgc
     5101 acccccacca agtggagggt cccctgccgg gtccccaaca tctactgaag aaggcaccag
     5161 cgaatccgca acgcccgagt caggccctgg tacctccaca gaaccatctg aaggtagtgc
     5221 gcctggttcc ccagctggaa gccctacttc caccgaagaa ggcacgtcaa ccgaaccaag
     5281 tgaaggatct gcccctggga ccagcactga accatctgag gccaagaaca caatcaccaa
     5341 gacactgaag ctgagaatcg tgcggcctta caacagcgcc gaggtggaaa aaatcgtggc
     5401 cgatgagaag aacaacagag agaaaatcgc tctggaaaaa aataaggaca aggttaaaga
     5461 agcctgctcc aagcacctga aagtggctgc ctactgcacc acccaggtcg agagaaacgc
     5521 ctgtctgttc tgcaaggcca gaaagctgga cgacaagttc taccaaaagc tgagaggcca
     5581 gttccccgac gccgtgttct ggcaggagat tagcgagatt ttcagacagc tgcagaagca
     5641 ggccgccgag atctacaacc agagcctgat cgagctgtac tacgagatct ttatcaaagg
     5701 caaaggaatt gccaacgctt ctagcgtgga acactacctg agcagagtgt gctaccggag
     5761 agccgccgag ctgttcaaga atgccgctat cgccagcgga ctgcggagca agatcaagtc
     5821 taacttccgg ctgaaggaac tcaagaatat gaaatcccgg ctgccaacaa caaagagcga
     5881 caactttcca atccccctgg tgaagcagaa aggaggccag tataccggct ttgagatcag
     5941 caaccacaac tctgatttca tcatcaagat cccattcggc agatggcaag tgaagaagga
     6001 aatcgacaag tacagacctt gggagaagtt cgacttcgag caggtgcaga agagtcctaa
     6061 gcctatcagc ctgctgctgt ctacccagag acggaaacgg aacaaaggct ggtccaagga
     6121 cgagggcacc gaagccgaaa tcaagaaggt gatgaacggc gactaccaga ccagctacat
     6181 cgaagtgaag aggggcagca agatcggcga gaagtctgcc tggatgctga acctgagcat
     6241 cgatgttcct aagatcgaca aaggagtgga ccccagcatc atcggcggaa ttgccgtggg
     6301 cgtgagaagc cctctggtct gtgccatcaa caatgctttt agcagataca gcatctccga
     6361 taacgacctg tttcacttta acaagaagat gttcgccagg cggagaatcc tgcttaaaaa
     6421 gaaccgccac aagagagccg gccacggcgc caagaacaag ctcaaaccta tcaccatcct
     6481 gaccgagaag agcgagcggt tccggaaaaa gctgatcgag agatgggcct gtgaaatcgc
     6541 cgacttcttc atcaaaaata aggtgggcac agtgcagatg gaaaacctgg aaggcatgaa
     6601 gagaaaggaa gatagctatt tcaacatccg gctgagaggc ttctggcctt acgccgagat
     6661 gcagaacaag atcgagttca agctgaagca atacggcatc gagatcagaa aggtggctcc
     6721 taacaacacc agcaaaacct gtagcaagtg cggccatctg aacaactact tcaacttcga
     6781 gtacagaaag aagaacaagt tcccccactt caagtgcgag aagtgcaact tcaaggaaaa
     6841 cgccgcttat aatgctgcgc tgaatatctc taatcctaag ctgaaatcta caaaggagga
     6901 acccggcggt ggcggaggga tggatgctaa gtcactaact gcctggtccc ggacactggt
     6961 gaccttcaag gatgtatttg tggacttcac cagggaggag tggaagctgc tggacactgc
     7021 tcagcagatc gtgtacagaa atgtgatgct ggagaactat aagaacctgg tttccttggg
     7081 ttatcagctt actaagccag atgtgatcct ccggttggag aagggagaag agcccggaag
     7141 cggatccgat tacaaagatg acgatgacaa agactacaag gatgatgatg ataagggatc
     7201 cggcgcaaca aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc
     7261 gatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     7321 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     7381 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     7441 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     7501 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     7561 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     7621 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     7681 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     7741 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     7801 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     7861 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     7921 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     7981 aacgcgttaa gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     8041 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     8101 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     8161 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     8221 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     8281 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     8341 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     8401 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag t
//
