LOCUS       sequence-247050-        7761 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ampicillin resistant multiplexing backbone with CRY2HA and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7761)
  AUTHORS   Kim JH, Rege M, Valeri J, Dunagin MC, Metzger A, Titus KR, Gilgenast
            TG, Gong W, Beagan JA, Raj A, Phillips-Cremins JE
  TITLE     LADL: light-activated dynamic looping for endogenous gene expression
            control.
  JOURNAL   Nat Methods. 2019 Jun 24. pii: 10.1038/s41592-019-0436-5. doi: 
            10.1038/s41592-019-0436-5.
  PUBMED    31235883
REFERENCE   2  (bases 1 to 7761)
  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: "Nat 
            Methods. 2019 Jun 24. pii: 10.1038/s41592-019-0436-5. doi: 
            10.1038/s41592-019-0436-5."
COMMENT     Sequence Label: S12.2_Bridge + Target
FEATURES             Location/Qualifiers
     source          1..7761
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      139..594
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(226..245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     436..457
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(611..630)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     730..752
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(790..808)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        876..980
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(981..1049,1050..1841)
                     /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(1199..1218)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2012..2600
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2501..2520
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2662..2910
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2662..2682
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2833..2852
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2929..3004
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(3090..3395)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     3245..3267
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3259..3276
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3260..3276
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3286..3305
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3286..3304
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3352..3372)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3352..3370)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3391..3407)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3391..3407)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3404..3426)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3415..3431
                     /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(3439..3445,3446..3463,3464..3469))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3484..3505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3843..4054
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          4055..4993
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4950..4970
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5030..5077
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6578..6598
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6629..6655
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6677..6730
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6731..7438
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(6873..6891)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7463..7480)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7469..7676
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   join(7685..7761,1..64)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(7685..7761,1..53)
                     /label=AAV2 ITR
ORIGIN
        1 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
       61 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
      121 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      181 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
      241 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      301 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
      361 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
      421 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
      481 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
      541 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
      601 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
      661 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
      721 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
      781 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
      841 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      901 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      961 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1021 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1081 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1141 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1201 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     1261 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     1321 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     1381 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     1441 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     1501 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     1561 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     1621 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     1681 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     1741 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1801 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1861 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1921 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1981 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2041 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2101 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     2161 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2221 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2281 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2341 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2401 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2461 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2521 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2581 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2641 gctggccttt tgctcacatg tgagggccta tttcccatga ttccttcata tttgcatata
     2701 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
     2761 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2821 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2881 ttatatatct tgtggaaagg acgaaacacc gggtcttcga gaagacctgt tttagagcta
     2941 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     3001 gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtttttagc
     3061 gcgtgcgcca attctgcaga caaatggctc tagagaccgg cgccgctaca gggcgcgtcc
     3121 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     3181 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     3241 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     3301 tagggcgaat tgggtaccgg gccccccctc gaggtcctcc agcttttgtt ccctttagtg
     3361 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3421 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3481 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccaccggtg
     3541 gtctcttaga ggtacccgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     3601 acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat tgacgtcaat
     3661 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     3721 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt gcccagtaca
     3781 tgaccttatg ggactttcct acttggcagt acatctacgt aaatactagt gtgcccgtca
     3841 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
     3901 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
     3961 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
     4021 tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg
     4081 gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca
     4141 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg
     4201 cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg
     4261 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc
     4321 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa
     4381 tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc
     4441 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat
     4501 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg
     4561 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag
     4621 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga
     4681 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc
     4741 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg
     4801 gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga
     4861 gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc
     4921 cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt
     4981 tcttccattt caggtgtcgt gaaagcttgg taccggatcc gccaccatga aaaggccggc
     5041 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagaag atggacaaaa agacgattgt
     5101 gtggtttaga cgtgaccttc gcattgagga caaccctgcg ttggcagcgg cagcgcatga
     5161 aggttcggta ttccctgtct ttatctggtg tcccgaagag gaggggcaat tctatcccgg
     5221 tcgcgcgtcc cgatggtgga tgaaacagtc gctcgcacat cttagccagt cgttgaaggc
     5281 gctcggctct gatctgacgc tcatcaaaac acacaataca atttctgcca ttctggattg
     5341 cattagggtg actggtgcca ccaaagttgt gttcaatcac ctttatgatc ccgtaagcct
     5401 ggtcagggat catacagtaa aagagaaact tgtagagagg ggaatcagcg tccaatcgta
     5461 caatggggac ttgttgtatg aaccgtggga aatctattgt gagaaaggca aacctttcac
     5521 ttccttcaat tcgtactgga agaaatgcct cgacatgtcc atcgagtcag tcatgctgcc
     5581 gccaccttgg agacttatgc ccattactgc tgcggcagag gcgatctggg cttgcagcat
     5641 cgaagaactt ggtctggaaa acgaagcgga aaaacccagt aacgcgctcc tgacgcgagc
     5701 gtggtcgcca ggctggtcga atgctgataa gctccttaat gagtttatcg agaaacagtt
     5761 gatcgactat gcgaagaata gcaagaaggt ggtgggaaat tccacttccc tcctgtcgcc
     5821 gtatctccac tttggggaga ttagcgtgag gcatgtgttc caatgtgcgc ggatgaagca
     5881 aatcatttgg gctcgcgaca aaaactctga aggagaggag tctgccgatt tgtttttgag
     5941 aggaatcggc cttagggagt attcacgata catctgcttc aactttccgt ttacacatga
     6001 acaatccctc ctcagccacc tccgcttctt tccgtgggac gcggacgtgg acaaattcaa
     6061 ggcctggcgg caggggcgca cgggataccc gttggtggat gcgggtatga gagagctgtg
     6121 ggcaacgggt tggatgcaca accgtattcg agtcatcgtt agtagctttg ccgttaagtt
     6181 cctcttgttg ccctggaaat gggggatgaa atacttttgg gacacgcttc tggatgcgga
     6241 cttggagtgc gatattcttg ggtggcagta catttcgggt tccatccctg acgggcatga
     6301 acttgaccga ttggataacc ctgcactgca aggagcaaag tacgacccgg agggagaata
     6361 catcaggcag tggctcccag agctggcccg tctgcccacc gagtggattc accacccgtg
     6421 ggatgctccc ctgaccgtcc ttaaagcctc aggggtcgaa ttggggacga attacgcgaa
     6481 acccatcgtc gatattgaca ccgcacgaga attgctggcg aaagccatct caagaacacg
     6541 cgaagcgcag attatgatcg gagcagcgcc ggctagcccc aagaagaaga gaaaggtgga
     6601 ggccagcggt tccggacggg ctgtatacta cccgtacgac gttccggact acgctctgtt
     6661 taaactgggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa
     6721 tcctggccca gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg
     6781 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     6841 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     6901 cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt
     6961 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
     7021 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     7081 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     7141 ccccgtaatg cagaagaaaa ccatgggctg ggaggcctcc tccgagcgga tgtaccccga
     7201 ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta
     7261 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta
     7321 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     7381 gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaaga
     7441 attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     7501 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     7561 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     7621 gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg
     7681 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     7741 gaggccgggc gaccaaaggt c
//
