LOCUS       Exported                7643 bp ds-DNA     circular SYN 08-AUG-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7643)
  AUTHORS   Institute for Molecular Bioscience
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Nov 8, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     EGFP Region has 6 mutations but it expresses just fine.1. aa65 Thr 
            to Ser2. aa66 Tyr to His3. aa68 Val to Val - this is fine4. aa145 
            Tyr to Phe5. aa163 Val to Ala6. aa198 Asn to Ser
FEATURES             Location/Qualifiers
     source          1..7643
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..821
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    772..792
                     /label=CMV -F
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1063..1081
                     /label=SV HIV-1 Forward 2
     misc_feature    complement(1169..1186)
                     /label=SV HIV-1 REV
     misc_feature    1686..1919
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    1748..1765
                     /label=SV RRE Reverse
     misc_feature    2233..2252
                     /label=RRE_3' [FW]
     misc_feature    2446..2563
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    2544..2563
                     /label=cPPT/CTS FW
     promoter        2618..2931
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     misc_feature    2850..2870
                     /label=mU6-F
     misc_feature    2929..2931
                     /label=POSITION FOR INSERTING sHRNA (KD)
     misc_feature    2932..3006
                     /label=hCavin1 shRNA
     primer_bind     3008..3024
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3066..3099
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      3688..3697
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3694..4413
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    3717..3734
                     /label=SV EGFP F1
     misc_feature    complement(3739..3760)
                     /label=EGFP-N
     misc_feature    complement(4000..4019)
                     /label=EXFP-R
     misc_feature    4347..4368
                     /label=EGFP-C
     misc_feature    complement(4387..4404)
                     /label=SV EGFP Reverse 2
     protein_bind    4441..4474
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    4530..5120
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     misc_feature    4530..4549
                     /label=SV WPRE Forward
     misc_feature    complement(4530..4549)
                     /label=hk_WPRE-R
     CDS             complement(5001..5012)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    5074..5092
                     /label=SV WPRE Forward 2
     primer_bind     complement(5123..5139)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             5649..5829
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5891..6479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(5971..5990)
                     /label=SV pBR322ori-R
     misc_feature    5971..5990
                     /label=SV ORI FORWARD
     misc_feature    6172..6190
                     /label=SV ORI MID FORWARD
     CDS             complement(6650..7510)
                     /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"
     misc_feature    7273..7292
                     /label=AMP-F
     misc_feature    7273..7292
                     
     misc_feature    complement(7335..7355)
                     
     promoter        complement(7511..7615)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    complement(7592..7612)
                     /label=SV AmpR Promoter Reverse 2
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga
     1561 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1621 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1681 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1741 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1801 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1861 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1921 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1981 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     2041 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     2101 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     2161 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     2221 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     2281 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     2341 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     2401 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2581 cagcagagat ccagtttggt tagtaccgcg ctctagagat ccgacgccgc catctctagg
     2641 cccgcgccgg ccccctcgca cagacttgtg ggagaagctc ggctactccc ctgccccggt
     2701 taatttgcat ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat
     2761 aatctgttct ttttaatact agctacattt tacatgatag gcttggattt ctataagaga
     2821 tacaaatact aaattattat tttaaaaaac agcacaaaag gaaactcacc ctaactgtaa
     2881 agtaattgtg tgttttgaga ctataaatat cccttggaga aaagccttgt ttcaccttcc
     2941 acgtcaagaa gatccgcgag gttcaagaga cctcgcggat cttcttgacg tggaaggtgt
     3001 tttttctcga ggtcgacggt atcgataagc tcgcttcacg agattccagc aggtcgaggg
     3061 acctaataac ttcgtatagc atacattata cgaagttata ttaagggttc caagcttaag
     3121 cggccgctga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc
     3181 aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta ggaacagaga
     3241 gacagcagaa tatgggccaa caaggatatc tgtggtaagc agttcctgcc ccggctcagg
     3301 gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag agaaccatca
     3361 gatgtttcca gggtgcccca aggacctgaa aatgaccctg tgccttattt gaactaacca
     3421 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc
     3481 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtat
     3541 tcccaataaa gcctcttgct gtttgcatcc gaatcgtgga ctcgctgatc cttgggaggg
     3601 tctcctcaga ttgattgact gcccacctcg ggggtctttc atcgaattct gcagtcgacg
     3661 gtaccgcggg cccgggatcc accggtcgcc accatggtga gcaagggcga ggagctgttc
     3721 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3781 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3841 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgag ccacggcgtc
     3901 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3961 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     4021 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     4081 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac
     4141 aacgtctata tcatggccga caagcagaag aacggcatca aggcgaactt caagatccgc
     4201 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     4261 ggcgacggcc ccgtgctgct gcccgacagc cactacctga gcacccagtc cgccctgagc
     4321 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     4381 atcactctcg gcatggacga gctgtacaag taacctgcag gcaattcgtc gagggaccta
     4441 ataacttcgt atagcataca ttatacgaag ttatacatgt ttaagggttc cggttccact
     4501 aggtacaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     4561 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4621 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4681 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4741 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4801 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     4861 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     4921 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     4981 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcgcggcct
     5041 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     5101 cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta gaaaaacatg
     5161 gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac
     5221 aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt
     5281 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     5341 ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact
     5401 tccctgattg gcagaactac acaccagggc cagggatcag atatccactg acctttggat
     5461 ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga
     5521 acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat
     5581 tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg
     5641 actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     5701 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     5761 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     5821 tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5881 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5941 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6001 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6061 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6121 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6181 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6241 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6301 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6361 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6421 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6481 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6541 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6601 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6661 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6721 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6781 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6841 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6901 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6961 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7021 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7081 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7141 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7201 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7261 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7321 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7381 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7441 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7501 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7561 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7621 ttccccgaaa agtgccacct gac
//
