LOCUS       Exported                5345 bp ds-DNA     circular SYN 17-JAN-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 5345)
  AUTHORS   Mikhail Shapiro
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, May 9, 2024 from SnapGene 5.1.7
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5345
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    585..644
                     /label=T7 insert
     promoter        585..605
                     /label=CMV_promoter
     primer_bind     619..654
                     /label=p101_T7m52_CMV_ClCAP
     primer_bind     complement(621..644)
                     /label=p69_R_linear_m52_T7
     primer_bind     623..664
                     /label=p70_F_5CMVUTR_T7_v2
     primer_bind     626..664
                     /label=p45_F_CMV5UTR_HA_T7
     primer_bind     626..664
                     /label=p55_F_T7_CMV_ClCapAG
     primer_bind     626..664
                     /label=p71_F_5CMVUTR_m34T7
     primer_bind     626..662
                     /label=p73_F_IVT_m34T7_5CMV
     promoter        626..644
                     /label=T7 prom
                     /note="/Transferred_From=pCMV-T7-Cas9 (MLM3613), 
                     pCMV-T7-Cas9
                     delta NcoI
                     /Transferred_Similarity=100.00%
                     /X-Transferred_From=pCRII-TOPO
                     /X-Transferred_Similarity=100.00%"
     5'UTR           645..814
                     /label=CMV 5'UTR
     promoter        725..746
                     /label=SR-alpha promoter
                     /note="pLannotate"
                     /note="/database=addgene"
                     /note="/fragment=True"
                     /note="/identity=100.0"
                     /note="/match_length=3.5830618892508146"
                     /note="/other=promoter"
     promoter        747..765
                     /label=SP6 promoter
                     /note="pLannotate"
                     /note="/database=addgene"
                     /note="/fragment=False"
                     /note="/identity=100.0"
                     /note="/match_length=100.0"
                     /note="/other=promoter"
     primer_bind     788..808
                     /label=p37_F_SEMPER_BB_ORF1_seqprim
     primer_bind     790..829
                     /label=p35_F_TTT_sfGFP2_HA_CMV173_v1
     primer_bind     790..823
                     /label=p33_F_ACC_sfGFP2_HA_CMV173
     primer_bind     complement(790..814)
                     /label=p_R_5UTR_SEMPER_BB
     primer_bind     complement(790..814)
                     /label=p_R_SEMPER_BB
     primer_bind     798..835
                     /label=p23_F_ORF1_sfGFP_CCCmutACC
     primer_bind     798..835
                     /label=p27_F_ORF1_sfGFP_CCCmutTTT
     primer_bind     809..832
                     /label=p62_F_TTT_sfGFP_HA_pZAG_HindIII
     primer_bind     809..830
                     /label=p64_F_ACC_sfGFP_HA_pZAG_HindIII
     primer_bind     816..841
                     /label=p25_F_v2_ORF1_sfGFP_CCCmutACC
     CDS             818..1537
                     /codon_start=1
                     /label=sfGFP(no ATGs)
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHIKRHDFFKSALPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHAVLL
                     EFVTAAGITHGKDELYK"
     primer_bind     818..841
                     /label=p29_F_v2_ORF1_sfGFP_CCCmutTTT
     primer_bind     complement(938..958)
                     /label=p38_R_sfGFP2(del5M)_seqprim
     primer_bind     complement(1514..1552)
                     /label=p34_R_sfGFP_HA_ACCeBFP2
     CDS             1517..1528
                     /codon_start=1
                     /product="KDEL ER retention tag"
                     /label=ER Retention
                     /translation="KDEL"
     primer_bind     1546..1566
                     /label=p23_F_ORF1_sfGFP_CCCmutACC
     primer_bind     1546..1561
                     /label=p64_F_ACC_sfGFP_HA_pZAG_HindIII
     primer_bind     1547..1572
                     /label=p25_F_v2_ORF1_sfGFP_CCCmutACC
     CDS             1549..2268
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSKLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     CDS             1549..2264
                     /label=EBFP2
                     /note="pLannotate"
                     /note="/database=fpbase"
                     /note="/fragment=False"
                     /note="/identity=99.6"
                     /note="/match_length=100.0"
                     /note="/other=CDS"
     primer_bind     1549..1572
                     /label=p29_F_v2_ORF1_sfGFP_CCCmutTTT
     primer_bind     1549..1566
                     /label=p27_F_ORF1_sfGFP_CCCmutTTT
     primer_bind     1549..1563
                     /label=p62_F_TTT_sfGFP_HA_pZAG_HindIII
     primer_bind     1549..1560
                     /label=p35_F_TTT_sfGFP2_HA_CMV173_v1
     primer_bind     complement(2244..2298)
                     /label=p63_R_EBFP2_HA_pZAG30_XhoI
     primer_bind     complement(2244..2276)
                     /label=p72_R_EBFP2_HA_m34Xeno
     primer_bind     complement(2257..2298)
                     /label=p65_R_IRES_mCherry_HA_pZAG30_XhoI
     misc_feature    2273..2274
                     /label=random
     primer_bind     2275..2293
                     /label=p68_F_linear_m52_XhoI
     misc_feature    2275..2280
                     /label=XhoI
     misc_feature    2281..2569
                     /label=hybrid UTR3
     primer_bind     complement(2552..2679)
                     /label=p54_R_oZAG49_IVT_ultramer_pZAG30
     misc_feature    2570..2679
                     /label=polyA
     misc_feature    2686..2734
                     /label=synthetic polyA
     misc_feature    2686..2734
                     /label=SPA
     polyA_signal    2789..2910
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2959..2975)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2983..2999
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     promoter        complement(3007..3037)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3052..3073
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3361..3949)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4120..4980)
                     /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(4981..5085)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggtcaga tcgcctggag
      661 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac
      721 tctagcctag gcttttgcaa aaagctattt aggtgacact atagaaggta cgcctgcagg
      781 taccgagctc ggatccagta gtttaaaccc cttcaccatg gtgtccaagg gcgaggaact
      841 gttcaccggc gtggtgccca tcctggtgga actggacggc gacgtgaacg gccacaagtt
      901 cagcgtgaga ggcgagggcg aaggcgacgc cacaaacgga aagctgaccc tgaagttcat
      961 ctgcaccacc ggcaagctgc ccgtgccttg gcctaccctc gtgaccacac tgacctacgg
     1021 cgtgcagtgc ttcagcagat accccgacca catcaagaga cacgatttct tcaagagcgc
     1081 cctgcccgag ggctacgtgc aggaacggac catcagcttc aaggacgacg gcacctacaa
     1141 gacaagagcc gaagtgaagt tcgagggcga caccctcgtg aaccggatcg agctgaaggg
     1201 catcgacttc aaagaggacg gcaacatcct gggccacaag ctggagtaca acttcaacag
     1261 ccacaacgtg tacatcaccg ccgacaagca gaaaaacggc atcaaagcca acttcaagat
     1321 ccggcacaac gtggaggacg gcagcgtgca gctggccgac cactaccagc agaacacccc
     1381 catcggagac ggccccgtgc tgctgcccga caaccactac ctgagcacac aaagcaagct
     1441 gagcaaggac cccaacgaga agcgggacca cgccgtgctg ctggaatttg tgaccgccgc
     1501 tggcatcacc cacggcaagg acgagctgta caagtgaccc gggttaccat ggtgtccaag
     1561 ggcgaggaac tgttcaccgg cgtggtgccc atcctggtgg aactggatgg cgacgtgaac
     1621 ggccacaagt tctctgtgcg gggagagggc gaaggcgacg ccacaaatgg caagctgacc
     1681 ctgaagttca tctgcaccac cggcaagctg cccgtgcctt ggcctaccct cgtgaccaca
     1741 ctgtctcacg gcgtgcagtg cttcgccaga taccccgacc acatgaagca gcacgatttc
     1801 ttcaagagcg ccatgcccga gggctacgtg caggaacgga ccatcttctt caaggacgac
     1861 ggcacctaca agaccagagc cgaagtgaag ttcgagggcg acaccctcgt gaaccggatc
     1921 gagctgaagg gcgtggactt caaagaggac ggcaacatcc tgggccacaa gctggagtac
     1981 aacttcaaca gccacaacat ctacatcatg gccgtgaagc agaaaaacgg catcaaagtg
     2041 aacttcaaga tccggcacaa cgtggaagat ggcagcgtgc agctggccga ccactaccag
     2101 cagaacaccc ccatcggaga tggccccgtg ctgctgcctg atagccacta cctgagcacc
     2161 cagagcaagc tgagcaagga ccccaacgag aagcgggacc acatggtgct gctggaattt
     2221 cggaccgccg ctggcatcac cctgggcatg gatgagctgt acaagtgata atacctcgag
     2281 ctggtactgc atgcacgcaa tgctagctgc ccctttcccg tcctgggtac cccgagtctc
     2341 ccccgacctc gggtcccagg tatgctccca cctccacctg ccccactcac cacctctgct
     2401 agttccagac acctcccaag cacgcagcaa tgcagctcaa aacgcttagc ctagccacac
     2461 ccccacggga aacagcagtg attaaccttt agcaataaac gaaagtttaa ctaagctata
     2521 ctaaccccag ggttggtcaa tttcgtgcca gccacaccct ggagctagca aaaaaaaaaa
     2581 aaaaaaaaaa aaaaaaaaag catatgacta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     2641 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaag agaccaataa aagatcttta
     2701 ttttcattag atctgtgtgt tggttttttg tgtgggctgg atgatcctcc agcgcgggga
     2761 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     2821 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2881 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     2941 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3001 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3061 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3121 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3181 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3241 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3301 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3361 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3421 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3481 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3541 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3601 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3661 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3721 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3781 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3841 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3901 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3961 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4021 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4081 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4141 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4201 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4261 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4321 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4381 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4441 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4501 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4561 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4621 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4681 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4741 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4801 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4861 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4921 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4981 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5041 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5101 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     5161 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     5221 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     5281 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgtacggg ccagatatac
     5341 gcgtt
//
