LOCUS       Exported                6408 bp DNA     circular SYN 15-JUN-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6408)
  AUTHORS   Matthew Millette
  TITLE     Direct Submission
  JOURNAL   Exported Mar 4, 2022 from SnapGene Viewer 6.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6408
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2192..2233
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..34
                     /label=terminator bridge -- single stranded
     misc_feature    7..11
                     /label=KpnI remnant
     misc_feature    12..810
                     /label=CBh promoter
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        299..576
                     /label=chicken beta-actin promoter
     intron          577..805
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     786..815
                     /label=bridge gap -- single stranded
     misc_feature    811..816
                     /label=ageI
                     /note="for CBh promoter"
     misc_feature    817..847
                     /label=4
     primer_bind     840..870
                     /label=42f
     misc_feature    843..847
                     /label=Kozak Sequence
     misc_feature    843..847
                     /label=NotI
     misc_feature    848..851
                     /label=Kozak Sequence
     CDS             852..1568
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     1549..1579
                     /label=terminator bridge -- single stranded
     primer_bind     complement(1549..1571)
                     /label=43r
     primer_bind     1580..1599
                     /label=IRES cODC OE  F
     misc_feature    1602..2191
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)
                     Includes M-Loop and Preferred, rather than Minimal AUG.
                     
                     Originally from Addgene plasmid - pMIGR AMPK KD (#27296)
                     
                     
                     "
     primer_bind     complement(2165..2184)
                     /label=IRES 670 OE R
     primer_bind     complement(2171..2202)
                     /label=30R
     primer_bind     2177..2267
                     /label=31U
     primer_bind     2179..2208
                     /label=bridge gap -- single stranded
     CDS             2192..2233
                     /codon_start=1
                     /label=G protein subunit alpha i2 (GNAI2)
                     /translation="GCTVSAEDKAAAER"
     CDS             2234..2248
                     /codon_start=1
                     /label=linker
                     /translation="GSGSG"
     primer_bind     2241..2273
                     /label=32F
     CDS             2255..2947
                     /codon_start=1
                     /product="codon optimized (rat)
                     Different enough that mt2 can't be aligned to it
                     "
                     /label=scarlet-I
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     2928..2986
                     /label=Primer 2
     primer_bind     complement(2928..2960)
                     /label=33R
     primer_bind     2932..2986
                     /label=FIXIT
     primer_bind     2933..2973
                     /label=34F
     misc_feature    2954..2959
                     /label=SphI
     misc_feature    2960..3069
                     /label=miRE 5'
     primer_bind     complement(2961..2981)
                     /label=ultramer amp R
     primer_bind     3055..3087
                     /label=original 97mer F
     primer_bind     3055..3087
                     /label=97mer forward
     primer_bind     3060..3087
                     /label=RF miRE Forward
     misc_feature    3070..3166
                     /label=97mer
     misc_feature    3087..3088
                     /label=mismatches
     misc_feature    3089..3109
                     /label=guide
     ncRNA           3112..3126
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     misc_feature    3129..3150
                     /label=MIMAT0000039 target
     primer_bind     complement(3152..3188)
                     /label=original 97mer R
     primer_bind     complement(3152..3188)
                     
     primer_bind     complement(3152..3174)
                     /label=RF miRE Rev
     misc_feature    3167..3283
                     
     misc_feature    3253..3258
                     /label=polyA signal
     misc_feature    3268..3273
                     /label=polyA signal
     primer_bind     complement(3284..3308)
                     /label=miRE reverse
     primer_bind     complement(3284..3308)
                     /label=miRE reverse N1 anneal
     primer_bind     3321..3369
                     /label=23F
     primer_bind     complement(3321..3350)
                     /label=35R
     primer_bind     complement(3330..3349)
                     /label=miRE rework blunt SphI R
     misc_feature    3349
                     /label=NotI Remainder
     primer_bind     complement(3432..3451)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_site      3497..3552
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     primer_bind     complement(3551..3570)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3911..3927)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3911..3927)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3924..3946)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3935..3951
                     /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(3959..3989)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4004..4025
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4084..4279
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4130..4265
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4192..4211
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4215..4226)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4285..4419
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4322..4341)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4376..4395
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4487..4504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4658..5246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4738..4757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5417..6277)
                     /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     6040..6059
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6278..6382)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(6385..6408,1..34)
                     /label=Primer 1
ORIGIN
        1 gtcgacgtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      421 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg
      601 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
      661 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa
      721 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca
      781 cctgcctgaa atcacttttt ttcaggttgg accggtcggc cgcagcccta tactgggctg
      841 cggccgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
      901 tcgagctgga cggcgacgta aacggccaca agttcagcgt ccgcggcgag ggcgagggcg
      961 atgccaccaa cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1021 cctggcccac cctcgtgacc accttcggct acggcgtggc ctgcttcagc cgctaccccg
     1081 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1141 gcaccatctc tttcaaggac gacggtacct acaagacccg cgccgaggtg aagttcgagg
     1201 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1261 tcctggggca caagctggag tacaacttca acagccacta cgtctatatc acggccgaca
     1321 agcagaagaa ctgcatcaag gctaacttca agatccgcca caacgttgag gacggcagcg
     1381 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1441 ccgacaacca ctacctgagc catcagtcca agctgagcaa agaccccaac gagaagcgcg
     1501 atcacatggt cctgctggag ttcgtgaccg ccgccgggat tacacatggc atggacgagc
     1561 tgtacaagta gcctgcaggg tcacctaaat aattccgccc ccccctctcc ctcccccccc
     1621 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     1681 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     1741 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     1801 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     1861 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     1921 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     1981 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     2041 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     2101 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     2161 aaaacacgat gataatatgg ccacaaccat gggctgcacc gtgagcgccg aggacaaggc
     2221 ggcggccgag cgcggctctg gttccggcac tagtgtgagt aaaggagagg ccgttataaa
     2281 agagttcatg cggttcaagg tacacatgga gggatcaatg aacggacacg agttcgaaat
     2341 cgaaggggaa ggagagggca ggccctatga gggtactcag accgccaaac tcaaagtgac
     2401 aaagggaggc cctttgcctt tctcttggga catcttgtca ccacaattta tgtatggctc
     2461 ccgagcattt ataaaacacc cagcagacat acccgattac tacaagcagt ctttcccaga
     2521 aggttttaaa tgggaaaggg tcatgaactt tgaggatggt ggtgctgtga cggtgacgca
     2581 ggatacttct ttggaggacg gaacactcat ctacaaggta aagctcaggg gcacgaactt
     2641 cccgccggac ggaccagtca tgcagaaaaa gactatggga tgggaggctt ccaccgaacg
     2701 gttgtatccc gaggacggtg ttttgaaggg agatataaaa atggctcttc ggctgaaaga
     2761 tggaggcaga tacttggcag attttaagac cacgtataag gcaaaaaaac cagtgcaaat
     2821 gccgggagca tataacgttg accgcaaact ggacatcacc tcacataacg aggattatac
     2881 agtagtagaa cagtacgaaa gaagcgaagg gaggcacagc accgggggta tggatgagct
     2941 gtataagtga taggcatgct gtttgaatga ggcttcagta ctttacagaa tcgttgcctg
     3001 cacatcttgg aaacacttgc tgggattact tcgacttctt aacccaacag aaggctcgag
     3061 aaggtatatt gctgttgaca gtgagcgcac tctttctagg aggttgtgat agtgaagcca
     3121 cagatgtatc acaacctcct agaaagagta tgcctactgc ctcggacttc aaggggctag
     3181 aattcgagca attatcttgt ttactaaaac tgaatacctt gctatctctt tgatacattt
     3241 ttacaaagct gaattaaaat ggtataaatt aaatcacttt tttcaattga cgcgtgatct
     3301 aattctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgg ccgcactcct
     3361 caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa
     3421 taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     3481 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     3541 ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag
     3601 tatttggttt agagtttggc aacatatgcc atatgctggc tgccatgaac aaaggtggct
     3661 ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa
     3721 agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac
     3781 atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct cctgactact
     3841 cccagtcata gctgtccctc ttctcttatg aagatccctc gacctgcagc ccaagcttgg
     3901 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3961 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4021 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga
     4081 tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4141 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4201 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4261 aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa
     4321 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4381 ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc
     4441 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4501 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4561 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4621 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4681 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4741 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4801 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac
     4861 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4921 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4981 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5041 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     5101 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5161 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5221 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5281 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5341 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5401 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5461 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5521 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5581 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5641 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5701 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5761 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5821 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5881 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5941 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6001 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6061 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6121 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6181 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6241 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6301 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6361 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctg
//
