LOCUS       CMV_AcrIIA4_cpGR        6514 bp    DNA     circular SYN 06-AUG-2025
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6514)
  AUTHORS   SnapGene License
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2025 from SnapGene 8.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6514
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        134..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        514..717
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        762..780
                     /note="T7 promoter
                     promoter for bacteriophage T7 RNA polymerase"
     CDS             806..826
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             845..1033
                     /codon_start=1
                     /label=AcrIIA4(Delta-N64/Q65/E66)_AsLOV2 (Y67)
                     /translation="MNINDLIREIKNKDYTVKLSGTDSNSITQLIIRVNNDGNEYVISE
                     SENESIVEKFISAFKNGW"
     CDS             1043..1255
                     /codon_start=1
                     /label=AncGR2 C176
                     /label=AncGR2\C176
                     /note="/vntifkey=21
                     after Rihtar et al. 2023 
                     ('>https://www.nature.com/articles/s41589-022-01136-x)"
                     /translation="NSSQNWQRFYQLTKLLDSMHEMVGGLLQFCFYTFVNKSLSVEFPE
                     MLAEIISNQLPKFNAGSVKPLLFHQK"
     CDS             1259..1303
                     /codon_start=1
                     /label=(GGS)5 Linker
                     /translation="GGSGGSGGSGGSGGS"
     CDS             1307..1831
                     /codon_start=1
                     /label=AncGR2N175
                     /note="/vntifkey=21
                     after Rihtar et al. 2023 
                     ('>https://www.nature.com/articles/s41589-022-01136-x)"
                     /translation="LISLLEVIEPEVLYSGYDSTLPDTSTRLMSTLNRLGGRQVVSAVK
                     WAKALPGFRNLHLDDQMTLLQYSWMSLMAFSLGWRSYKQSNGNMLCFAPDLVINEERMQ
                     LPYMYDQCQQMLKISSEFVRLQVSYDEYLCMKVLLLLSTVPKDGLKSQAVFDEIRMTYI
                     KELGKAIVKREG"
     CDS             1841..1906
                     /codon_start=1
                     /label=AcrIIA4(Delta-N64/Q65/E66)_AsLOV2 (Y67)
                     /translation="YEDEEEFYNDMQTITLKSELN"
     polyA_signal    2013..2237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2283..2711
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2725..3054
                     /note="SV40 promoter
                     SV40 enhancer and early promoter"
     rep_origin      2905..3040
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3121..3915
                     /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"
     polyA_signal    4089..4210
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4259..4275)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4283..4299
                     /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(join(4307..4313,4314..4331,4332..4337))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4352..4373
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4661..5246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5417..6208,6209..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"
     promoter        complement(6278..6382)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
       61 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
      121 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      541 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      601 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      661 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      721 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
      781 gagacccaag ctggctagca tggccccgaa gaaaaagcga aaggtgggcg gaagcggagg
      841 ttccatgaac attaacgacc tgattcggga aataaagaac aaggactaca cggtcaagct
      901 gtcaggcacc gactccaact ctataacaca gctcattata agggttaata atgatggcaa
      961 cgaatatgtc atctctgaat ccgagaatga atcaatcgtg gagaaattca tcagtgcatt
     1021 taaaaacggc tggggtccgg ggaacagcag ccagaactgg cagaggttct accagctgac
     1081 caagctgctg gacagcatgc acgagatggt gggcggcctg ctccagttct gcttctacac
     1141 cttcgtgaac aagagcctga gcgtggagtt ccccgagatg ctggccgaga tcatcagcaa
     1201 ccagctgcct aagtttaatg ccggcagcgt gaagcccctg ctgttccacc agaagggggg
     1261 cggaagcggc ggatccggcg gatctggcgg atcgggcggg agtggactga tcagcctgct
     1321 ggaggtgatc gagcccgagg tgctgtacag cggctacgac agcaccctgc ccgacaccag
     1381 caccaggctg atgagcaccc tgaacaggct gggcggcagg caggtggtga gcgccgtgaa
     1441 gtgggccaag gccctgcccg gcttcaggaa cctgcacctg gacgaccaga tgaccctgct
     1501 gcagtacagc tggatgagcc tgatggcctt cagcctgggc tggaggagct acaagcagag
     1561 caacggcaac atgctgtgct tcgcccccga cctggtgatc aacgaggaga ggatgcagct
     1621 gccctacatg tacgaccagt gccagcagat gctgaagatc agcagcgagt tcgtgaggct
     1681 gcaggtgagc tacgacgagt acctgtgcat gaaggtgctg ctgctgctga gcaccgtgcc
     1741 caaggacggc ctgaagagcc aggccgtgtt cgacgagatc aggatgacct acatcaagga
     1801 gctgggcaag gccatcgtga agagggaggg cggtccgggg tacgaagatg aggaagagtt
     1861 ctacaatgat atgcaaacta tcacactgaa atctgagctg aactagctcg agcggccgcc
     1921 actgtgctgg atatctgcag aattccacca cactggacta gtggatccga gctcggtacc
     1981 aagcttaagt ttaaaccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2041 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2101 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2161 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2221 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2281 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2341 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2401 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2461 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2521 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2581 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2641 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2701 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2761 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2821 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2881 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2941 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3001 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3061 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     3121 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3181 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3241 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3301 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3361 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3421 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3481 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3541 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3601 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3661 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3721 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3781 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3841 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3901 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     3961 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4021 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4081 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4141 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4201 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4261 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4321 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4381 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4441 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4501 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4561 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4621 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4681 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4741 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4801 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4861 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4921 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4981 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5041 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5101 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5161 gctcttgatc cggcaaacaa accaccgctg gtagcggttt 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 gccacctgac gtcgacggat
     6421 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     6481 gttaagccag tatctgctcc ctgcttgtgt gttg
//
