LOCUS       Exported                7531 bp ds-DNA     circular SYN 14-NOV-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7531)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Nov 24, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7531
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     142..158
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     182..219
                     /label=Primer 1
     misc_feature    202..1226
                     /label=sali arm
     CDS             1164..1226
                     /codon_start=1
                     /translation="CAVGTTGQKSISRVIEYLEHC"
     primer_bind     complement(1210..1246)
                     /label=sali neo 3'
     CDS             1233..1256
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1266..1586
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1593..1619
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1620..2333
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2343..2399
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2400..3194
                     /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))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     3181..3220
                     /label=Primer 3
     misc_feature    3201..4943
                     /label=kpni arm
     primer_bind     complement(3201..3220)
                     /label=gRNA
     misc_feature    3201..3202
                     /label=mut for gRNA
     primer_bind     complement(4926..4978)
                     /label=Primer 4
     promoter        complement(5034..5052)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5073..5089)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5097..5113
                     /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(5121..5151)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5166..5187
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5475..6063)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6234..7094)
                     /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(7095..7199)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      121 ggttttccca gtcacgacgt tgtaaaacga cggccagtgc cactagtggc cgcggcggcc
      181 gcagcttgcc tgcaggtcga ctaggtaagt gaatgtggac tggaaatgac gtatgaattt
      241 tggggtcctt tgtagatgac tacgggatat gtagcagagc caacagcatc aagtgagtta
      301 cagtatcatt ttttaccagc ttgactgttt ttaatctgtt tcttcagatc aatgtaacta
      361 cagtaaggga atatcttcca gaaggagatt tctccataat gacagagatg cttaaaaagt
      421 tcttaagctt catgaatctt acggtaagta acttttattg tatattgcag tttatatgag
      481 tttttgatta ttttccagac aaagtcttaa cactgtagcc caggctggca atccacatgt
      541 ttcagccatt ggaccagtgg gacctggcta gtttatgtgt ttttcaattt aaatgtaata
      601 catcgaaggt gaaaataata atcccatagt gttgttcttc cctgtggcaa aatagttaac
      661 ataagcagct tacagaaggt acattttggt ttatagttct aaatttttgt gtgtgtgttt
      721 ttttttctgt ttgtttttgt ttttttgaga cagggtttct ctgtgtagcc ctggctgtcc
      781 tggaactcac cctgtagacc aggctggcct cgaactcaga aatccgcctg cttctgcctc
      841 tcaagtgcta ggatcaaagg cgtgcgccac caccaccagg ctagttctaa aatttttagt
      901 catgtgtggc aagagcagcg tggaagagca gagtggccag ctaatagaga atgcctgtgt
      961 atctgctttc tccttttatt ccagctgtgc ttctaggctg tgggatgatg ccacccacaa
     1021 acagaaaact attgtggcag aatgttatat tactaaggca atgatttaat ttcagtgtac
     1081 tctttactac gtagactttg atactttttt ttgtttacca ttttgttaag tattaactct
     1141 aatttctctc cttcccctct tagtgtgctg ttggaacaac aggccagaag tctatctcta
     1201 gagtgattga atatttggaa cattgcgtcg acgattacaa ggatgacgat gacaagggtt
     1261 ccggcggcgt gcaggtggag actatctccc caggagacgg gcgcaccttc cccaagcgcg
     1321 gccagacctg cgtggtgcac tacaccggga tgcttgaaga tggaaagaaa gttgattcct
     1381 cccgggacag aaacaagccc tttaagttta tgctaggcaa gcaggaggtg atccgaggct
     1441 gggaagaagg ggttgcccag atgagtgtgg gtcagagagc caaactgact atatctccag
     1501 attatgccta tggtgccact gggcacccag gcatcatccc accacatgcc actctcgtct
     1561 tcgatgtgga gcttctaaaa ctggaagtcg attatccgta cgacgtacca gactacgcag
     1621 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1681 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1741 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1801 tgaccacctt cggctacggc ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc
     1861 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1921 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1981 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2041 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2101 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2161 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2221 tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2281 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggatccg
     2341 gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt cgaagagaat cctggaccga
     2401 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     2461 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     2521 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     2581 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     2641 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     2701 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     2761 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     2821 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     2881 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     2941 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3001 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3061 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3121 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     3181 acgagttctt ctgaggtacc ttgcttcagg tgcttggtaa tgctgaagct atccttagac
     3241 aaagaaaaga catgaaagaa gtccttgaag atataccaag aacattcatc agtatcattc
     3301 attcgtgttt ggatttttat gatcacccga tttcttcatc atgcattctg cgtttgctaa
     3361 atgacagtta ctacatcaat ctgcagctat caaaaatgag ggaaaaggtt caggctgtta
     3421 acaatcccat gcagtattta aatacactta cttggcagag tctatacctg cccctgtttc
     3481 cttgctgtat ttcaggcaag gtttaaagag ggcaatttgt gctgctgtta gtgcaactgc
     3541 tgtgtatgtt gagccactgt tgtcatgcca gtcaggtgca aaggcagctt agctactgag
     3601 gtattgaatg ttctgaggac attctagaca acagcttaga tcctttttca ggctcatttg
     3661 cttttgcttt ttgttgaatg attccaatcg taaataaagc ttttaataat tttgtgaatt
     3721 ttttggttgt tgttccctga actactgtct atatttaaaa tgagatggaa tccaaagaca
     3781 caagggatta atagtatatt tttttattct tgattaggtt tgggtgttga actatttttc
     3841 acttctgaga ccatgaccat attcaatgtc ataccattat gtgtcatagc tataggcaca
     3901 agaaaacaaa tagcagtttg agggaatatt atgttatata agataatgtg ccctgttaaa
     3961 ggattaagca aaaatagaca aacccagggt agtttacact taatgctggg gaggctcttg
     4021 aaacattgtt acgttttgag ggacgttctc tagatatatt ttctaatgtt cagttcagta
     4081 aatataataa ggaagctaaa acaccaatgt ggaatccgtt tttccagata gcgtgtatat
     4141 tcttctatag agtgacagga tcaattgcat aagcgcaaag ccttaaaatt gctggtgtag
     4201 agaagaccct tttttattca gattctatgt tcatagagca attgtttaga aaatggttgt
     4261 ggaacacaca gaacatggtt tatagggtaa atttcataga ttgttttttc tttttttttt
     4321 tttctttttt ttttttgtat tgttcagacc actggttata cattttctcc tttttttatt
     4381 aattggcctg aggacaggct ttccagagat gtttcattaa tacttttaat tacctttcag
     4441 atagttacat catgtttctt cattggattt gtaaaactta aagccataaa aatattagtt
     4501 tggtgtgtat tggggaaaat agctaaaagt ctaactttga ctcattttag actttgttat
     4561 ttccttgtat aaagtaacaa accggggctc ttgtatccgt gccagctgta atgtttttaa
     4621 atgcagtggc tgccttctat tgtcttccta tttttgataa tgcagattgt tgggaaatct
     4681 ataaggaagt aactgactct aggcaaattg ttttcttcct cctacccgcc caacccccta
     4741 cccgtccgtc accttttagg aacacagtat gccagtgtaa tgtgggaacc attgagactg
     4801 tatttgccct gagtaccaaa ctaatttagt gaattttttt ttaaacatac tggtaaatga
     4861 tgtccacaaa tttccttaga tatattttaa ttaattgtct taaaatacat tgatatttat
     4921 taatcattgg atttggggac agaggtaccg ggccccccct cgaggtcgat cgacggtatc
     4981 gataagcttg atctagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag
     5041 tgagggttaa ttccgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5101 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5161 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5221 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5281 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5341 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5401 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5461 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5521 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5581 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5641 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5701 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5761 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5821 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5881 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     5941 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6001 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6061 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6121 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6181 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6241 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6301 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6361 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6421 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6481 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6541 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6601 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6661 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6721 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6781 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6841 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6901 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6961 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7021 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7081 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7141 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7201 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     7261 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     7321 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     7381 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     7441 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     7501 agatgcgtaa ggagaaaata ccgcatcagg c
//
