LOCUS       sequence-396674-        4936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for cloning iSBH-sgRNAs .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4936)
  AUTHORS   Pelea O, Mayes S, Ferry QRV, Fulga TA, Sauka-Spengler T
  TITLE     Specific Modulation of CRISPR Transcriptional Activators through 
            RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos
  JOURNAL   bioRxiv 2023.05.08.539738
REFERENCE   2  (bases 1 to 4936)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2023.05.08.539738"
COMMENT     Sequence Label: pcDNA3.1_SV40-iBlue-pA
FEATURES             Location/Qualifiers
     source          1..4936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        255..503
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     255..275
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     426..445
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        522..597
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      686..1114
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(773..792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     983..1004
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1123..1143)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1128..1457
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1308..1443
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1370..1389
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1504..2463
                     /codon_start=1
                     /product="naturally monomeric infrared fluorescent protein 
                     derived from a truncated bacteriophytochrome (Yu et al., 
                     2015)"
                     /label=mIFP
                     /translation="MSVPLTTSAFGHAFLANCEREQIHLAGSIQPHGILLAVKEPDNVV
                     IQASINAAEFLNTNSVVGRPLRDLGGDLPLQILPHLNGPLHLAPMTLRCTVGSPPRRVD
                     CTIHRPSNGGLIVELEPATKTTNIAPALDGAFHRITSSSSLMGLCDETATIIREITGYD
                     RVMVVRFDEEGNGQILSERRRADLEAFLGNRYPASTIPQIARRLYEHNRVRLLVDVNYT
                     PVPLQPRISPLNGRDLDMSLSCLRSMSPCHQKYMQDMGVGATLVCSLMVSGRLWGLIAC
                     HHYEPRFVPFHIRAAGEALAETCAIRIATLESFAQSQSK"
     polyA_signal    2609..2730
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2646..2665)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2700..2719
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2779..2795)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2779..2795)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2792..2814)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2803..2819
                     /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(2827..2833,2834..2851,2852..2857))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2872..2893
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3010..3027)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3181..3769)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3261..3280)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3940..4731,4732..4800))
                     /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     4563..4582
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4801..4905)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagtgagggc ctatttccca tgattccttc atatttgcat atacgataca
      301 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
      361 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      421 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
      481 tcttgtggaa aggacgaaac accgggtctt cgagaagacc tgttttagag ctagaaatag
      541 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      601 tttgctagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
      661 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
      721 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
      781 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      841 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      901 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
      961 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1021 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1081 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     1141 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     1201 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     1261 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     1321 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     1381 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     1441 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcgcc
     1501 accatgtcgg taccgctgac tacctcagca ttcggccacg cgtttctggc taactgtgaa
     1561 cgcgagcaga tccacctggc gggctccatt cagccgcacg gtatcctgct ggctgtgaaa
     1621 gagccggaca acgtggtgat ccaggcttct attaacgctg cggagttcct gaacaccaac
     1681 tctgttgttg gccgtccgct gcgtgacctg ggcggcgatc tgcctttgca gatcctgccg
     1741 cacctgaacg gcccgctgca cctggctccg atgaccctgc gttgtaccgt gggttctccg
     1801 ccgcgtcgtg tggactgtac cattcatcgt ccgtctaacg gcggcctgat cgtagaactg
     1861 gaaccagcaa ccaagaccac taacattgcg ccggctctgg acggtgcgtt tcatcgtatc
     1921 acttcttcat cctccctgat gggcctgtgt gacgaaaccg cgactattat ccgtgagatt
     1981 actggctacg accgtgtgat ggtagtacgt ttcgatgaag agggtaatgg ccaaattctg
     2041 tccgaacgtc gtcgtgcgga cctggaagcg ttcctgggta accgctaccc ggcgtctact
     2101 attccgcaga tcgctcgtcg cctgtacgaa cataaccgtg ttcgcctgct ggtagatgtg
     2161 aactatactc cggttccgct acagccgcgc atcagcccgc tgaacggtcg tgatctggat
     2221 atgtccctgt cttgcctgcg ctctatgtcc ccgtgccacc agaaatacat gcaggacatg
     2281 ggcgttggcg cgaccctggt ttgctctctg atggtgtctg gtcgtctgtg gggtctgatc
     2341 gcttgccacc actacgaacc gcgcttcgtt ccgttccaca ttcgcgctgc tggcgaagcg
     2401 ctggcggaaa cttgtgcgat ccgcatcgcg acgctggaga gctttgcaca gtctcagtcc
     2461 aaatgatgta caagtaaagc acgtgctacg agatttcgat tccaccgccg ccttctatga
     2521 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     2581 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     2641 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2701 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     2761 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     2821 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2881 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2941 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3001 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3061 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3121 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3181 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3241 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3301 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3361 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3421 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3481 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3541 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3601 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3661 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3721 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3781 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3841 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3901 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3961 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4021 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4081 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4141 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4201 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4261 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4321 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4381 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4441 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4501 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4561 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4621 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4681 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4741 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4801 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4861 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4921 agtgccacct gacgtc
//
