LOCUS       pDest_U6_A2_CG2_        8246 bp DNA     circular SYN 25-JUN-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8246)
  AUTHORS   dacer
  TITLE     Direct Submission
  JOURNAL   Exported Mar 3, 2023 from SnapGene Viewer 6.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8246
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     promoter        17..121
                     /gene="bla"
                     /label=AmpR promoter
     CDS             122..982
                     /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"
     primer_bind     complement(340..359)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1153..1741
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1642..1661
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1895..1912
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2029..2050
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2065..2095
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2103..2119
                     /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     2108..2130
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2127..2143
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2162..2182
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2164..2182
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2219..2235
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2219..2235
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        2792..3689
                     /label=cmlc2 promoter
     CDS             3690..4409
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3735..3756)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3996..4015)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4343..4364
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(4435..4452)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4493..4511)
                     /label=pCEPF
                     /note="2 sites!"
     polyA_signal    4711..4845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4791..4809
                     /label=pCEPF
                     /note="2 sites!"
     primer_bind     4925..4948
                     /label=zU6-3 -285
                     /note="counting starts at the 5' end of the primer"
     primer_bind     5100..5118
                     /label=U6-3_seq2
                     /note="ordered this one"
     promoter        5110..5209
                     /label=zU6-3 -100 to 0
     primer_bind     5129..5147
                     /label=U6-3_seq1
     misc_RNA        5232..5305
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5318..5334
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5342..5465
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        5490..5520
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5574..6254
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(5640..5659)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             6574..6879
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     6758..6777
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(6920..7044)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7063..7079)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8079..8100
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
       61 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
      121 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
      181 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
      241 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
      301 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
      361 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
      421 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
      481 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
      541 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
      601 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
      661 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
      721 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
      781 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
      841 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
      901 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
      961 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     1021 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     1081 gaccaaaatg ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1141 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1201 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1261 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     1321 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     1381 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     1441 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     1501 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     1561 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     1621 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     1681 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     1741 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     1801 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     1861 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     1921 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     1981 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     2041 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     2101 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     2161 cgcaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc
     2221 tagaactagt ggatctgctg ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa
     2281 gtacttgagt aattttactt gattactgta cttaagtatt atttttgggg atttttactt
     2341 tacttgagta caattaaaaa tcaatacttt tacttttact taattacatt tttttagaaa
     2401 aaaaagtact ttttactcct tacaatttta tttacagtca aaaagtactt attttttgga
     2461 gatcacttca ttctattttc ccttgctatt accaaaccaa ttgaattgcg ctgatgccca
     2521 gtttaattta aatgttattt attctgccta tgaaaatcgt tttcacatta tatgaaattg
     2581 gtcagacatg ttcattggtc ctttggaagt gacgtcatgt cacatctatt accacaatgc
     2641 acagcacctt gacctggaaa ttagggaaat tataacagtc aatcagtgga agaaaatgga
     2701 ggaagtatgt gattcatcag cagctgcgag cagcacagtc caaaatcagc cacaggatca
     2761 agagcacccg tggccgtatc ttcgcagatc taaagcttaa atcagttgtg ttaaataaga
     2821 gacattcaaa ataaatgtaa atgagctctc caaatcagca gacttaacat tctttaaaat
     2881 gattgattca atagtgataa aaatcaggca tagccagttg taactttaga taaattacag
     2941 aaaatgtcaa atacagagaa ccgattcttt tttatgatac atccaagcac acatttaaca
     3001 caatccaggc aaaccccgaa tttcacagtc acaagcactg tttgtacaag agctttgcct
     3061 aaggacacac agtctctata agtccaggtc gttggtttca ctcttatttt aaacatgtga
     3121 catttttcct gccatcctgt cttaggctgc tgtttgcttc attccatgtc acattaaatt
     3181 cctcagtagc accttttaca cacacagcca atcttttcca gaaaattcaa ttgctttgaa
     3241 gagataatgt gtgaacaaat ccatttagaa aaggaaaatt aagaatttgt aaaatcatct
     3301 gtaaattgtt ggcattcttc tgtatatgaa catcacatca tttacaggta aaggtctggt
     3361 cattaattat atgacaattt actggtatta ttttgtgaaa ggggctattt tcaatgcgtt
     3421 catccatcct tttcatccct caaatctctc attcacgtcc ccctccccat ctgcacactt
     3481 tatctcattt ccaccctgct ggaatctgag cacttgtgca gttatcaggg ctcctgtatt
     3541 taggaggctc tgggtgtcca tgtaggggac gaacagaaac actgcagacc tttatagaag
     3601 aacaattgat aagagtcctc atacataaag actccattag taagccagtg acccaggagc
     3661 ccagaccaac agcaaagcag acagtgacca tggtgagcaa gggcgaagag ctgttcaccg
     3721 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     3781 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     3841 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     3901 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3961 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     4021 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     4081 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     4141 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     4201 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     4261 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     4321 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     4381 ctctcggcat ggacgagctg tacaagtaaa gcggcccctc tcgagcctct agaactatag
     4441 tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     4501 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4561 tgtaaccatt ataagctgca ataaacaaga gatctgatct agaggatcat aatcagccat
     4621 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     4681 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     4741 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4801 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcatcat cgatgcgtct
     4861 tttgttctgg tcatcaagga ggggggaatg ttctgcgcat gcctgtgggg gagggagaag
     4921 gacacgtcac tgaaaacgtc cctgcatcac accgagacac ccaatcactc aagccgagac
     4981 cagataattt tgcatatgct ttaaagtttg aaaaatacca cggtaaaccc tcacacaaac
     5041 tctggatttg agatctttca ggttttatca gtttgcaggt ttatgtcacc atgatatagg
     5101 gtcagacttg atctaaggga gctgaataag tggtttagtc actcaccacc tcccaaaaac
     5161 atacccagaa gtccctggta tatatagctc tccctccagc tcttggttcg gagctcgccc
     5221 gccgctcggg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     5281 tgaaaaagtg gcaccgagtc ggtgcttttt tggtaccgta aaacgacggc cagtgaatta
     5341 tcaactatgt ataataaagt tgaacgagaa acgtaaaatg atataaatat caatatatta
     5401 aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca
     5461 ctatggcggc cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg
     5521 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
     5581 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     5641 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     5701 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     5761 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
     5821 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     5881 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     5941 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     6001 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     6061 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     6121 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     6181 gaatgcttaa tgaattacaa cagtactgcg atgagtggca ggcggggcgt aatctagagg
     6241 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     6301 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     6361 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     6421 atatctccgg ttcggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     6481 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     6541 gctcctttgc cgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     6601 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     6661 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt cccccgtgaa
     6721 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     6781 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     6841 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     6901 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt
     6961 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     7021 ttcaactttt ctatacaaag ttgatagctt ggcgtaatca tggtcatagc tgtttcctgc
     7081 tcgagggccc atctggcctg tgtttcagac accagggagt ctctgctcac gtttcctgct
     7141 atttgcagcc tctctatcaa gactaataca cctcttcccg catcggctgc ctgtgagagg
     7201 cttttcagca ctgcaggatt gcttttcagc cccaaaagag ctaggcttga cactaacaat
     7261 tttgagaatc agcttctact gaagttaaat ctgaggtttt acaactttga gtagcgtgta
     7321 ctggcattag attgtctgtc ttatagtttg ataattaaat acaaacagtt ctaaagcagg
     7381 ataaaacctt gtatgcattt catttaatgt tttttgagat taaaagctta aacaagaatc
     7441 tctagttttc tttcttgctt ttacttttac ttccttaata ctcaagtaca attttaatgg
     7501 agtacttttt tacttttact caagtaagat tctagccaga tacttttact tttaattgag
     7561 taaaattttc cctaagtact tgtactttca cttgagtaaa atttttgagt actttttaca
     7621 cctctgtcaa gaactcctgg acaaacctct gacctgtgtg gaacagagtg gatatgggtg
     7681 tctgaacaga tattcacgtc ttttgcagat cagagggcat ttctggtgtt gtgcaatgac
     7741 ctgggtccaa caggtaggcg tactcgtatc caagagcatt gttggacagg aggtagtctc
     7801 cattcctata aagaggaagg aatggcacca tgtagtagcc gtcattgtgg ccaattggtg
     7861 cattggtacg tgggtagatg gaccggggag gctgatgagt tcttagccag cctttccccg
     7921 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     7981 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     8041 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     8101 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     8161 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     8221 attaacgctt acaatttagg tggcac
//
