LOCUS       Exported                5215 bp ds-DNA     circular SYN 15-NOV-2019
DEFINITION  pVC4 used as vector for AAV plasmid
            pVC22 used for the CMV EGFP NLS insert.
ACCESSION   .
VERSION     .
KEYWORDS    pVC 45 pAAV CMV EGFP NLS WPRE pA
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 5215)
  AUTHORS   Engel Lab
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jan 8, 2021 from SnapGene 3.2.1
            http://www.snapgene.com
COMMENT     PCR to produce CMV EGFP NLS insert:
            
            CloneAmp HiFi PCR Premix- 12.5ul
            CMV EGFP NLS Fwd Primer (10uM)- 0.75ul
            CMV EGFP NLS Rev Primer (10uM)- 0.75ul
            Template pVC22 (10ng/ul)- 2ul
            Water- 9ul
            --------------------
            Total- 25 ul.
            
            PCR Cycle:
            1. 95C-5min
            2. 98C- 10s
            3. 55C- 15s
            4. 72C-15s
            Step 2-4 for 32 cycles
            
            5. 72C- 5min
            Hold at 4C
            
            pVC4 double digest to produce vector backbone:
            
            Buffer 2.1- 5ul
            pVC4- 3ul
            HindIII-HF- 4ul
            Water- 38ul
            -----------------
            Total- 50ul
            
            Incubate 1 hour at 37C
            
            Add 0.54ul 5M NaCl to convert buffer 2.1 to buffer 3.1.
            
            Add 4ul BglII
            
            Incubate 1 hour at 37C.
            
            Run PCR product and digested pVC4 on 0.8% agarose gel.
            
            Cut bands: PCR product- 1174bp, digested plasmid- ~4000bp.
            
            Gel purify using NucleoSpin Gel and PCR Clean-Up Kit. 
            
            Nanodrop. PCR product- 183ng/ul, plasmid- 55ng/ul.
            
            Set up In-fusion reaction:
            
            EGFP PCR product (insert)- 100ng- 0.54ul
            pVC4 digested plasmid (vector)- 100ng- 1.8ul
            5X In-Fusion HD Enzyme Premix- 2ul
            Water- 5.66ul
            ----------------------------
            Total- 10ul
            
            Incubate at 50C for 15min
            Transfer sample to ice.
            
            Transform this into Stellar cells.
FEATURES             Location/Qualifiers
     source          1..5215
                     /organism="recombinant plasmid"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
     protein_bind    228..249
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        264..294
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    302..318
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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     326..342
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    379..509
                     /note="L-ITR"
     misc_feature    380..547
                     /note="ITR2"
     misc_feature    complement(381..547)
                     /note="3' ITR"
     promoter        570..773
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    796..876
                     /note="MCS"
                     /note="multiple cloning site"
     regulatory      878..887
                     /regulatory_class="other"
                     /note="Kozak sequence"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    878..883
                     /note="Kozak"
     CDS             884..1600
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="EGFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    995..1000
                     /note="Kozak"
     CDS             1616..1642
                     /codon_start=1
                     /product="nuclear localization signal of human c-myc (Dang 
                     and Lee, 1988)"
                     /note="c-myc NLS"
                     /translation="PAAKRVKLD"
     CDS             1643..1669
                     /codon_start=1
                     /product="nuclear localization signal of human c-myc (Dang 
                     and Lee, 1988)"
                     /note="c-myc NLS"
                     /translation="PAAKRVKLD"
     CDS             1670..1696
                     /codon_start=1
                     /product="nuclear localization signal of human c-myc (Dang 
                     and Lee, 1988)"
                     /note="c-myc NLS"
                     /translation="PAAKRVKLD"
     misc_feature    1724..2312
                     /note="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     misc_feature    1976..1981
                     /note="Kozak"
     misc_feature    2079..2097
                     /note="WPRE qPCR Forw"
     misc_feature    2099..2113
                     /note="WPRE Probe"
     misc_feature    2116..2137
                     /note="WPRE qPCR Rev"
     misc_feature    2153..2158
                     /note="Kozak"
     CDS             complement(2195..2206)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /note="Factor Xa site"
                     /translation="IEGR"
     primer_bind     complement(2315..2331)
                     /note="KS primer"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2361..2482
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     misc_feature    2606..2773
                     /note="ITR2"
     misc_feature    2606..2772
                     /note="3' ITR"
     misc_feature    2644..2774
                     /note="R-ITR"
     primer_bind     complement(2792..2808)
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      2950..3405
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     misc_feature    3407..3412
                     /note="Kozak"
     promoter        3431..3535
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             3536..4396
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4567..5155
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4737..4742
                     /note="Kozak"
     misc_feature    5094..5099
                     /note="Kozak"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
       61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      181 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      241 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      301 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccagattt
      361 aattaaggcc ttaattaggc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
      421 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
      481 agtggccaac tccatcacta ggggttcctt gtagttaatg attaacccgc catgctactt
      541 atctacgtag ccatgctcta ggaagatctg tgatgcggtt ttggcagtac atcaatgggc
      601 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      661 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      721 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
      781 tgaaccgtca gatccgctag cgctaccgga ctcagatctc gagctcaagc ttcgaattct
      841 gcagtcgacg gtaccgcggg cccgggatcc accggtcgcc accatggtga gcaagggcga
      901 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
      961 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     1021 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     1081 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1141 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     1201 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1261 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     1321 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     1381 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     1441 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     1501 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     1561 cgccgccggg atcactctcg gcatggacga gctgtacaag agatctcgag ctgatcctgc
     1621 tgccaaacgc gttaaactag accctgctgc caaacgcgtt aaactagacc ctgctgccaa
     1681 acgcgttaaa ctagactaaa gcggccgcga caagcttatc gataatcaac ctctggatta
     1741 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     1801 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     1861 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     1921 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     1981 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     2041 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     2101 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     2161 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     2221 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     2281 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gacccgggcg
     2341 gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     2401 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     2461 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     2521 gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga
     2581 taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact
     2641 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     2701 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     2761 agcgagcgcg cagccttaat taacctaatt cactggccgt cgttttacaa cgtcgtgact
     2821 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     2881 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     2941 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3001 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3061 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3121 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3181 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3241 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3301 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3361 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     3421 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3481 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3541 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3601 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3661 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3721 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3781 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3841 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3901 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3961 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4021 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4081 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4141 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4201 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4261 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4321 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4381 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4441 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4501 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4561 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4621 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4681 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4741 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4801 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4861 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4921 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4981 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5041 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5101 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5161 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatg
//
