LOCUS       Exported                7301 bp ds-DNA     circular SYN 01-JUN-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jun 11, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7301
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..861
                     /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"
     rep_origin      1032..1620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    1908..1929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        1944..1974
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1982..1998
                     /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)."
     primer_bind     2006..2022
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2043..2061
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2089..2315
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2316..2496
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2543..2668
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    3161..3394
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     promoter        3801..4041
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    4021..4026
                     /label=U6 TATA Box (-30 to -24)
     misc_feature    4029..4049
                     /label=Original human U6 promoter 5' end, -1 @ end
     misc_feature    4050
                     /label=U6 +1
     gap             4051..4070
                     /label=gRNA Target Sequence
                     /estimated_length=20
     misc_feature    4075
                     /label=F a-u flip
                     /note="Original gRNA sequence flipped A to a T, since
                     mammalian Pol-III termination sequence is TTTT "
     misc_feature    4083..4087
                     /label=E. stem extension
     misc_feature    4092..4096
                     /label=E. stem complementary
     misc_feature    4137..4156
                     /label=Constant 3' sgRNA scaffold
     misc_feature    4157..4162
                     /label=Pol-III Termination Sequence
                     /note="Sanger verified that the construct on hand is a T7
                     and not a T6, there is additional T after this labeled 
                     sequence. Gao et al 2018 found T6 is 99% efficient while T7
                     is 100% efficient."
     misc_feature    4204..4321
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        4390..4601
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor
                     EF-1-alpha"
     CDS             4627..5226
                     /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    5242..5830
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     CDS             complement(5713..5724)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5902..6135
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    5954
                     /label=+1 of Reverse Transcription
     polyA_signal    6213..6334
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      6374..6509
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(6530..6548)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6558..6574)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      6716..7171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7197..7301
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
       61 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      121 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
      181 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
      241 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
      301 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
      361 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
      421 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
      481 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
      541 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
      601 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
      661 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
      721 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
      781 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
      841 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
      901 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
      961 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1021 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1081 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1141 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     1201 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1261 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1321 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1381 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1441 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1501 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1561 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1621 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1681 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     1741 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     1801 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     1861 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     1921 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     1981 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
     2041 gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg
     2101 caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag
     2161 agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag
     2221 gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag
     2281 atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct
     2341 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     2401 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     2461 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     2521 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     2581 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     2641 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     2701 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     2761 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     2821 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     2881 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     2941 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     3001 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     3061 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     3121 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     3181 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     3241 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     3301 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     3361 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     3421 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     3481 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     3541 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     3601 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     3661 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     3721 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     3781 caaccccgag gggacccaga gagggcctat ttcccatgat tccttcatat ttgcatatac
     3841 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
     3901 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     3961 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     4021 tatatatctt gtggaaagga cgaaacaccg gcggctgccg gcggtgcccg gtttaagagc
     4081 tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt
     4141 ggcaccgagt cggtgctttt ttgaattcta gatcttgaga caaatggcag tattcatcca
     4201 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     4261 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     4321 tcgggtttat tacagggaca gcagagatcc actttggcgc cggctcgagt ggctccggtg
     4381 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     4441 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     4501 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     4561 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga cgcgggatcc
     4621 gccaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     4681 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     4741 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     4801 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     4861 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     4921 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     4981 aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt
     5041 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     5101 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     5161 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     5221 gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact
     5281 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     5341 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     5401 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     5461 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     5521 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     5581 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     5641 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     5701 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     5761 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     5821 gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact
     5881 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc
     5941 tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     6001 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     6061 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     6121 ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag tatttataac
     6181 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     6241 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     6301 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     6361 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     6421 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     6481 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
     6541 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     6601 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     6661 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     6721 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     6781 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     6841 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     6901 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     6961 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     7021 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     7081 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     7141 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     7201 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     7261 aaccctgata aatgcttcaa taatattgaa aaaggaagag t
//
