LOCUS       sequence-247048-        4184 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ampicillin resistant multiplexing backbone without CRY2HA and 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4184)
  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 4184)
  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: S12.1_Empty_Bridge
FEATURES             Location/Qualifiers
     source          1..4184
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      699..1287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1188..1207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1349..1597
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1349..1369
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1520..1539
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1616..1691
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(1777..2082)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     1932..1954
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1946..1963
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1947..1963
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1973..1992
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1973..1991
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2039..2059)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2039..2057)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2078..2094)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2078..2094)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2091..2113)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2102..2118
                     /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(2126..2132,2133..2150,2151..2156))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2171..2192
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2573..2590)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2579..2786
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2795..2935
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2795..2924
                     /label=AAV2 ITR
     rep_origin      3010..3465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3097..3116)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3307..3328
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3482..3501)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3601..3623
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3661..3679)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3747..3851
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3852..3920,3921..4184,1..528)
                     /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(4070..4089)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
       61 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
      121 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
      181 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
      241 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
      301 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
      361 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
      421 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
      481 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
      541 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
      601 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
      661 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
      721 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
      781 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
      841 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
      901 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
      961 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1021 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1081 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1141 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     1201 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     1261 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     1321 gccttttgct ggccttttgc tcacatgtga gggcctattt cccatgattc cttcatattt
     1381 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     1441 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     1501 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     1561 cttggcttta tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa gacctgtttt
     1621 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     1681 cgagtcggtg cttttttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     1741 ttttagcgcg tgcgccaatt ctgcagacaa atggctctag agaccggcgc cgctacaggg
     1801 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     1861 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     1921 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga
     1981 ctcactatag ggcgaattgg gtaccgggcc ccccctcgag gtcctccagc ttttgttccc
     2041 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2101 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2161 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2221 accggtggtc tcttagaggt acccgttaca taacttacgg taaatggccc gcctggctga
     2281 ccgcccaacg acccccgccc attgacgtca atagtaacgc caatagggac tttccattga
     2341 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     2401 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattgtgcc
     2461 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatg tatccgcatc
     2521 tgatgctacc gtggttgagt tagcgtcgga attcctagag ctcgctgatc agcctcgact
     2581 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2641 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2701 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2761 gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat ggagttggcc
     2821 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     2881 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     2941 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     3001 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3061 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     3121 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3181 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     3241 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3301 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     3361 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     3421 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     3481 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     3541 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3601 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     3661 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     3721 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3781 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3841 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3901 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3961 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4021 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4081 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4141 agaatgactt ggttgagtac tcaccagtca cagaaaagca tctt
//
