LOCUS       Exported                6412 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 6412)
  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..6412
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2196..2237
                     /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..850
                     /label=13
     primer_bind     843..874
                     /label=42f
     misc_feature    851..855
                     /label=Kozak Sequence
     CDS             856..1572
                     /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     1553..1583
                     /label=terminator bridge -- single stranded
     primer_bind     complement(1553..1575)
                     /label=43r
     primer_bind     1584..1603
                     /label=IRES cODC OE  F
     misc_feature    1606..2195
                     /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(2169..2188)
                     /label=IRES 670 OE R
     primer_bind     complement(2175..2206)
                     /label=30R
     primer_bind     2181..2271
                     /label=31U
     primer_bind     2183..2212
                     /label=bridge gap -- single stranded
     CDS             2196..2237
                     /codon_start=1
                     /label=G protein subunit alpha i2 (GNAI2)
                     /translation="GCTVSAEDKAAAER"
     CDS             2238..2252
                     /codon_start=1
                     /label=linker
                     /translation="GSGSG"
     primer_bind     2245..2277
                     /label=32F
     CDS             2259..2951
                     /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     2932..2990
                     /label=Primer 2
     primer_bind     complement(2932..2964)
                     /label=33R
     primer_bind     2936..2990
                     /label=FIXIT
     primer_bind     2937..2977
                     /label=34F
     misc_feature    2958..2963
                     /label=SphI
     misc_feature    2964..3073
                     /label=miRE 5'
     primer_bind     complement(2965..2985)
                     /label=ultramer amp R
     primer_bind     3059..3091
                     /label=original 97mer F
     primer_bind     3059..3091
                     /label=97mer forward
     primer_bind     3064..3091
                     /label=RF miRE Forward
     misc_feature    3074..3170
                     /label=97mer
     misc_feature    3091..3092
                     /label=mismatches
     misc_feature    3093..3113
                     /label=guide
     ncRNA           3116..3130
                     /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    3133..3154
                     /label=MIMAT0000039 target
     primer_bind     complement(3156..3192)
                     /label=original 97mer R
     primer_bind     complement(3156..3192)
                     
     primer_bind     complement(3156..3178)
                     /label=RF miRE Rev
     misc_feature    3171..3287
                     
     misc_feature    3257..3262
                     /label=polyA signal
     misc_feature    3272..3277
                     /label=polyA signal
     primer_bind     complement(3288..3312)
                     /label=miRE reverse
     primer_bind     complement(3288..3312)
                     /label=miRE reverse N1 anneal
     primer_bind     3325..3373
                     /label=23F
     primer_bind     complement(3325..3354)
                     /label=35R
     primer_bind     complement(3334..3353)
                     /label=miRE rework blunt SphI R
     misc_feature    3353
                     /label=NotI Remainder
     primer_bind     complement(3436..3455)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_site      3501..3556
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     primer_bind     complement(3555..3574)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3915..3931)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3915..3931)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3928..3950)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3939..3955
                     /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(3963..3993)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4008..4029
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4088..4283
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4134..4269
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4196..4215
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4219..4230)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4289..4423
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4326..4345)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4380..4399
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4491..4508)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4662..5250)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4742..4761)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5421..6281)
                     /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     6044..6063
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6282..6386)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(6389..6412,1..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 cgcgcacgcc tatactggcg
      841 tgcgcggccg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      901 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtccgcgg cgagggcgag
      961 ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1021 gtgccctggc ccaccctcgt gaccaccttc ggctacggcg tggcctgctt cagccgctac
     1081 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1141 gagcgcacca tctctttcaa ggacgacggt acctacaaga cccgcgccga ggtgaagttc
     1201 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1261 aacatcctgg ggcacaagct ggagtacaac ttcaacagcc actacgtcta tatcacggcc
     1321 gacaagcaga agaactgcat caaggctaac ttcaagatcc gccacaacgt tgaggacggc
     1381 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1441 ctgcccgaca accactacct gagccatcag tccaagctga gcaaagaccc caacgagaag
     1501 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggattacaca tggcatggac
     1561 gagctgtaca agtagcctgc agggtcacct aaataattcc gcccccccct ctccctcccc
     1621 cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat
     1681 gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt
     1741 cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt
     1801 gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc
     1861 gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc
     1921 acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat
     1981 agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc
     2041 ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg
     2101 tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct
     2161 ttgaaaaaca cgatgataat atggccacaa ccatgggctg caccgtgagc gccgaggaca
     2221 aggcggcggc cgagcgcggc tctggttccg gcactagtgt gagtaaagga gaggccgtta
     2281 taaaagagtt catgcggttc aaggtacaca tggagggatc aatgaacgga cacgagttcg
     2341 aaatcgaagg ggaaggagag ggcaggccct atgagggtac tcagaccgcc aaactcaaag
     2401 tgacaaaggg aggccctttg cctttctctt gggacatctt gtcaccacaa tttatgtatg
     2461 gctcccgagc atttataaaa cacccagcag acatacccga ttactacaag cagtctttcc
     2521 cagaaggttt taaatgggaa agggtcatga actttgagga tggtggtgct gtgacggtga
     2581 cgcaggatac ttctttggag gacggaacac tcatctacaa ggtaaagctc aggggcacga
     2641 acttcccgcc ggacggacca gtcatgcaga aaaagactat gggatgggag gcttccaccg
     2701 aacggttgta tcccgaggac ggtgttttga agggagatat aaaaatggct cttcggctga
     2761 aagatggagg cagatacttg gcagatttta agaccacgta taaggcaaaa aaaccagtgc
     2821 aaatgccggg agcatataac gttgaccgca aactggacat cacctcacat aacgaggatt
     2881 atacagtagt agaacagtac gaaagaagcg aagggaggca cagcaccggg ggtatggatg
     2941 agctgtataa gtgataggca tgctgtttga atgaggcttc agtactttac agaatcgttg
     3001 cctgcacatc ttggaaacac ttgctgggat tacttcgact tcttaaccca acagaaggct
     3061 cgagaaggta tattgctgtt gacagtgagc gcactctttc taggaggttg tgatagtgaa
     3121 gccacagatg tatcacaacc tcctagaaag agtatgccta ctgcctcgga cttcaagggg
     3181 ctagaattcg agcaattatc ttgtttacta aaactgaata ccttgctatc tctttgatac
     3241 atttttacaa agctgaatta aaatggtata aattaaatca cttttttcaa ttgacgcgtg
     3301 atctaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atggccgcac
     3361 tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca
     3421 caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct
     3481 tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa
     3541 ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa
     3601 tgagtatttg gtttagagtt tggcaacata tgccatatgc tggctgccat gaacaaaggt
     3661 ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata
     3721 gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt
     3781 taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac
     3841 tactcccagt catagctgtc cctcttctct tatgaagatc cctcgacctg cagcccaagc
     3901 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3961 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4021 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4081 cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4141 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4201 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4261 ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata atggttacaa
     4321 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4381 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca ttaatgaatc
     4441 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4501 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4561 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4621 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4681 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4741 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4801 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc
     4861 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4921 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4981 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5041 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5101 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5161 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5221 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5281 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5341 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5401 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5461 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5521 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5581 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5641 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5701 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5761 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5821 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5881 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5941 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6001 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6061 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6121 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6181 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6241 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6301 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6361 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tg
//
