LOCUS       Exported                7322 bp DNA     circular SYN 12-AUG-2020
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7322)
  AUTHORS   Walfred Tang
  TITLE     Direct Submission
  JOURNAL   Exported Mar 1, 2022 from SnapGene 6.0.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7322
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     misc_feature    complement(1..234)
                     /label=PB 3' TR
                     /note="color: #00ffff; direction: LEFT"
     enhancer        244..474
                     /locus_tag="core insul(1)"
                     /label=core insul enhancer
                     /label=Core Insulator(1)
                     /note="color: #8080ff; direction: RIGHT"
     primer_bind     473..493
                     /label=C33 113-F1-cF
                     /note="color: black; sequence: 
                     AAGGCCTCCACGGCCACTAGTAAGGATCTGCGATCGCTCCGG"
     primer_bind     474..491
                     /label=C372 261-F1-F
                     /note="color: black; sequence: AGGCCTCCACGGCCACTA; added: 
                     2019-11-26"
     promoter        494..734
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     494..514
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
                     /note="color: black; sequence: GAGGGCCTATTTCCCATGATT"
     misc_feature    744..785
                     /label=gRNA insertion site by Esp3I
                     /note="color: #ff9900; direction: BOTH"
     misc_feature    787..879
                     /label=gRNA scafford opti
                     /note="color: #00ccff; direction: RIGHT"
     primer_bind     complement(881..922)
                     /label=C373 261-F1-R
                     /note="color: black; sequence: 
                     CGATCGCAGATCCTTACTAGGTGATACGCCTAACTAGagcgc; added: 
                     2019-11-26"
     primer_bind     903..928
                     /label=C33 113-F1-cF
                     /note="color: black; sequence: 
                     AAGGCCTCCACGGCCACTAGTAAGGATCTGCGATCGCTCCGG"
     promoter        908..1153
                     /locus_tag="EF1\promoter"
                     /label=EF1a promoter
                     /note="color: #ffffff"
     misc_feature    933..1151
                     /label=Human EEF1A1 promoter
                     /note="chr6:73,521,000-73,521,229 (hg38)1st exon"
                     /note="color: #33cccc; direction: RIGHT"
     primer_bind     complement(987..1006)
                     /label=S60 EF1a-R
                     /note="color: black; sequence: GCACCCGTTCAATTGCCGAC; added:
                     2019-12-09"
     primer_bind     complement(1137..1161)
                     /label=C34 113-F1-cR
                     /note="color: black; sequence: gtggcTCGAGCTGTGTTCTGGCGGC"
     primer_bind     1142..1173
                     /label=C35 113-F2-cF
                     /note="color: black; sequence: 
                     CAGAACACAGCTCGAgccaccatgtctagact"
     CDS             1163..1909
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /note="color: #993366"
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(1340..1359)
                     /label=FS52 Tet3G.sR
                     /note="color: black; sequence: TGGGTATGATGCCTGTCCAG; added:
                     2019-12-09"
     primer_bind     1578..1597
                     /label=FS49 Tet3G.sF
                     /note="color: black; sequence: ACTTTACACTGGGCTGCGTA; added:
                     2019-12-09"
     misc_feature    1941..2527
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
                     /note="This feature has 3 segments:
                        1: 1941 .. 2515 / #a6acb3
                        2: 2516 .. 2518 / #a6acb3 / ATG
                        3: 2519 .. 2527 / #a6acb3"
     primer_bind     2030..2049
                     /label=S29  IRES2-F
                     /note="color: black; sequence: tattgccgtcttttggcaat"
     primer_bind     2030..2049
                     /label=S29 IRES2-F
                     /note="color: black; sequence: tattgccgtcttttggcaat; added:
                     2019-12-09"
     primer_bind     complement(2078..2097)
                     /label=S30 IRES2-R
                     /note="color: black; sequence: cctaggaatgctcgtcaaga; added:
                     2019-12-09"
     CDS             2528..3322
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /note="color: #ccffcc"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3329..3377
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
                     /note="color: #a6acb3"
     primer_bind     complement(3362..3408)
                     /label=C36 113-F2-cR
                     /note="color: black; sequence: 
                     GGGAAAAAGCCATACCAATGGGCCCggatccgaacaaacgacccaac"
     primer_bind     complement(3387..3406)
                     /label=S31 Ins-R
                     /note="color: black; sequence: GAAAAAGCCATACCAATGGG; added:
                     2019-12-09"
     enhancer        complement(3402..3632)
                     /locus_tag="core insul(1)"
                     /label=core insul enhancer
                     /label=Core Insulator(1)
                     /note="color: #8080ff; direction: LEFT"
     misc_feature    3633..3945
                     /label=PB 5' TR
                     /note="color: #00ffff; direction: RIGHT"
     primer_bind     complement(4432..4448)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     primer_bind     complement(4432..4448)
                     /label=S27 M13-R
                     /note="color: black; sequence: CAGGAAACAGCTATGAC; added: 
                     2019-12-09"
     protein_bind    4456..4472
                     /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)."
                     /note="color: #31849b"
     promoter        complement(4480..4510)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 4480 .. 4486 / #ffffff / -10
                        2: 4487 .. 4504 / #ffffff
                        3: 4505 .. 4510 / #ffffff / -35"
     protein_bind    4525..4546
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     rep_origin      complement(4834..5422)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(5593..6453)
                     /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: 5593 .. 6384 / #ccffcc
                        2: 6385 .. 6453 / #ccffcc / signal sequence
                     Cleavage site after base 6384"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6454..6558)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     rep_origin      complement(6584..7039)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     7180..7197
                     /label=S26 M13-F
                     /note="color: black; sequence: TGTAAAACGACGGCCAGT; added: 
                     2019-12-09"
     primer_bind     7181..7197
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
ORIGIN
        1 ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg tgtaaaattg
       61 acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa
      121 gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt
      181 tatttattta ttaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt
      241 atgagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
      301 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
      361 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
      421 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaggcctc
      481 cacggccact agtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      541 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
      601 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      661 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      721 cttgtggaaa ggacgaaaca ccggagacga tgcgtaggga gatccggaat ctattggccc
      781 gtctccgttt aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt
      841 atcaacttga aaaagtggca ccgagtcggt gcttttttta gcgctctagt taggcgtatc
      901 acctagtaag gatctgcgat cgctccggtg cccgtcagtg ggcagagcgc acatcgccca
      961 cagtccccga gaagttgggg ggaggggtcg gcaattgaac gggtgcctag agaaggtggc
     1021 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     1081 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     1141 ccagaacaca gctcgagcca ccatgtctag actggacaag agcaaagtca taaactctgc
     1201 tctggaatta ctcaatggag tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa
     1261 gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga
     1321 tgccctgcca atcgagatgc tggacaggca tcatacccac tcctgccccc tggaaggcga
     1381 gtcatggcaa gactttctgc ggaacaacgc caagtcatac cgctgtgctc tcctctcaca
     1441 tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct
     1501 ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc
     1561 tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg agcatcaagt
     1621 agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc tgaaacaagc
     1681 aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc tggaactaat
     1741 catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccgaccg acgcccttga
     1801 cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct
     1861 gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtaag gccgctcgat
     1921 aagcttgata tcgaattccg cccctctccc tccccccccc ctaacgttac tggccgaagc
     1981 cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct
     2041 tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt
     2101 ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct
     2161 ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc
     2221 ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag
     2281 gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc
     2341 tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga
     2401 tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt
     2461 ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc
     2521 cacaaccatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     2581 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     2641 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     2701 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     2761 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     2821 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     2881 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     2941 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     3001 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     3061 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     3121 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     3181 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     3241 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     3301 ccttcttgac gagttcttct gaggggatcg gcaataaaaa gacagaataa aacgcacggg
     3361 tgttgggtcg tttgttcgga tccgggccca ttggtatggc tttttccccg tatcccccca
     3421 ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc
     3481 ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg
     3541 accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac
     3601 atccctgggg gctttggggg ggggctgtcc ctgatatcta taacaagaaa atatatatat
     3661 aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc
     3721 ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca
     3781 aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg
     3841 agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca
     3901 agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaatctag ctgcatcagg
     3961 atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc tatctgggca
     4021 tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg aaagagtttg
     4081 ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc atgatgattc
     4141 gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc acctgggata
     4201 ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag cgcatcagca
     4261 acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt aatgacagcg
     4321 gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg ccgcagaaat
     4381 ggacatggat accccgtgag ttacccggcg ggcgcgcttg gcgtaatcat ggtcatagct
     4441 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4501 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4561 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4621 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4681 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4741 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4801 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4861 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4921 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4981 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5041 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5101 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5161 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5221 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     5281 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5341 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5401 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5461 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5521 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5581 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5641 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5701 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5761 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5821 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5881 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5941 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6001 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6061 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6121 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6181 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6241 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6301 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6361 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6421 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6481 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6541 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     6601 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     6661 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     6721 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     6781 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     6841 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     6901 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     6961 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     7021 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     7081 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     7141 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
     7201 cgcgcctcgt tcattcacgt ttttgaaccc gtggaggacg ggcagactcg cggtgcaaat
     7261 gtgttttaca gcgtgatgga gcagatgaag atgctcgaca cgctgcagaa cacgcagcta
     7321 ga
//
