LOCUS       sequence-390528-        7200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To create ""long homology arm"" (~1000bp) knock-in donor vectors for
            split-intein Gal4[21-881]C-int.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7200)
  AUTHORS   Ewen-Campen B, Luan H, Xu J, Singh R, Joshi N, Thakkar T, Berger B, 
            White BH, Perrimon N
  TITLE     split-intein Gal4 provides intersectional genetic labeling that is 
            repressible by Gal80.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Jun 13;120(24):e2304730120. doi: 
            10.1073/pnas.2304730120. Epub 2023 Jun 5.
  PUBMED    37276389
REFERENCE   2  (bases 1 to 7200)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-06-13"; volume: "120(24):e2304730120. 
            doi"; pages: " 10.1073/pnas.2304730120. Epub 2023 Jun 5"
COMMENT     Sequence Label: pHD-T2A-split-intein-Gal4[21-881]-C-int
FEATURES             Location/Qualifiers
     source          1..7200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(94..113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     304..325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             741..794
                     /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             3153..3497
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     protein_bind    3749..3782
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4008..4017
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4014..4691
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(4144..4162)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4194..4214)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4412..4431
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4606..4629
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4812..4933
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4849..4868)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4903..4922
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    4963..4996
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5015..5035)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5015..5033)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5054..5070)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5054..5070)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5067..5089)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5078..5094
                     /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(5102..5108,5109..5126,5127..5132))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5147..5168
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5285..5302)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5456..6044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5536..5555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6215..7006,7007..7075))
                     /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     6838..6857
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7076..7180)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
       61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      361 atttataagg gattttgccg atttcggcct attggttaaa aaaatgagct gatttaacaa
      421 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 caagcttcac gacctgaggc gcgccgggcc agtacccaaa aagcgggggg tccggtggaa
      721 gcggaggtag cgcggccgcc gagggccgcg gcagcctgct gacctgcggc gatgtggagg
      781 agaaccccgg gcccgctagc atgatgctga agaagatcct gaagatcgag gagctggatg
      841 agcgcgagct gatcgatatc gaggtgagcg gcaaccacct gttctacgcc aacgatatcc
      901 tgacccacaa ctgctccaaa gaaaaaccga agtgcgccaa gtgtctgaag aacaactggg
      961 agtgtcgcta ctctcccaaa accaaaaggt ctccgctgac tagggcacat ctgacagaag
     1021 tggaatcaag gctagaaaga ctggaacagc tatttctact gatttttcct cgagaagacc
     1081 ttgacatgat tttgaaaatg gattctttac aggatataaa agcattgtta acaggattat
     1141 ttgtacaaga taatgtgaat aaagatgccg tcacagatag attggcttca gtggagactg
     1201 atatgcctct aacattgaga cagcatagaa taagtgcgac atcatcatcg gaagagagta
     1261 gtaacaaagg tcaaagacag ttgactgtat cgattgactc ggcagctcat catgataact
     1321 ccacaattcc gttggatttt atgcccaggg atgctcttca tggatttgat tggtctgaag
     1381 aggatgacat gtcggatggc ttgcccttcc tgaaaacgga ccccaacaat aatgggttct
     1441 ttggcgacgg ttctctctta tgtattcttc gatctattgg ctttaaaccg gaaaattaca
     1501 cgaactctaa cgttaacagg ctcccgacca tgattacgga tagatacacg ttggcttcta
     1561 gatccacaac atcccgttta cttcaaagtt atctcaataa ttttcacccc tactgcccta
     1621 tcgtgcactc accgacgcta atgatgttgt ataataacca gattgaaatc gcgtcgaagg
     1681 atcaatggca aatccttttt aactgcatat tagccattgg agcctggtgt atagaggggg
     1741 aatctactga tatagatgtt ttttactatc aaaatgctaa atctcatttg acgagcaagg
     1801 tcttcgagtc aggttccata attttggtga cagccctaca tcttctgtcg cgatatacac
     1861 agtggaggca gaaaacaaat actagctata attttcacag cttttccata agaatggcca
     1921 tatcattggg cttgaatagg gacctcccct cgtccttcag tgatagcagc attctggaac
     1981 aaagacgccg aatttggtgg tctgtctact cttgggagat ccaattgtcc ctgctttatg
     2041 gtcgatccat ccagctttct cagaatacaa tctccttccc ttcttctgtc gacgatgtgc
     2101 agcgtaccac aacaggtccc accatatatc atggcatcat tgaaacagca aggctcttac
     2161 aagttttcac aaaaatctat gaactagaca aaacagtaac tgcagaaaaa agtcctatat
     2221 gtgcaaaaaa atgcttgatg atttgtaatg agattgagga ggtttcgaga caggcaccaa
     2281 agtttttaca aatggatatt tccaccaccg ctctaaccaa tttgttgaag gaacaccctt
     2341 ggctatcctt tacaagattc gaactgaagt ggaaacagtt gtctcttatc atttatgtat
     2401 taagagattt tttcactaat tttacccaga aaaagtcaca actagaacag gatcaaaatg
     2461 atcatcaaag ttatgaagtt aaacgatgct ccatcatgtt aagcgatgca gcacaaagaa
     2521 ctgttatgtc tgtaagtagc tatatggaca atcataatgt caccccatat tttgcctgga
     2581 attgttctta ttacttgttc aatgcagtcc tagtacccat aaagactcta ctctcaaact
     2641 caaaatcgaa tgctgagaat aacgagaccg cacaattatt acaacaaatt aacactgttc
     2701 tgatgctatt aaaaaaactg gccactttta aaatccagac ttgtgaaaaa tacattcaag
     2761 tactggaaga ggtatgtgcg ccgtttctgt tatcacagtg tgcaatccca ttaccgcata
     2821 tcagttataa caatagtaat ggtagcgcca ttaaaaatat tgtcggttct gcaactatcg
     2881 cccaataccc tactcttccg gaggaaaatg tcaacaatat cagtgttaaa tatgtttctc
     2941 ctggctcagt agggccttca cctgtgccat tgaaatcagg agcaagtttc agtgatctag
     3001 tcaagctgtt atctaaccgt ccaccctctc gtaactctcc agtgacaata ccaagaagca
     3061 caccttcgca tcgctcagtc acgccttttc tagggcaaca gcaacagctg caatcattag
     3121 tgccactgac cccgtctgct ttgtttggtg gcgccaattt taatcaaagt gggaatattg
     3181 ctgatagctc attgtccttc actttcacta acagtagcaa cggtccgaac ctcataacaa
     3241 ctcaaacaaa ttctcaagcg ctttcacaac caattgcctc ctctaacgtt catgataact
     3301 tcatgaataa tgaaatcacg gctagtaaaa ttgatgatgg taataattca aaaccactgt
     3361 cacctggttg gacggaccaa actgcgtata acgcgtttgg aatcactaca gggatgttta
     3421 ataccactac aatggatgat gtatataact atctattcga tgatgaagat accccaccaa
     3481 acccaaaaaa agagtaacct ctagagtcga ctaaagccaa atagaaatta ttcagttctg
     3541 gcttaagttt ttaaaagtga tattatttat ttggttgtaa ccaaccaaaa gaatgtaaat
     3601 aactaataca taattatgtt agttttaagt tagcaacaaa ttgattttag ctatattagc
     3661 tacttggtta ataaatagaa tatatttatt taaagataat tcgtttttat tgtcagggag
     3721 tgagtttgct taaaaactcg tttggatcat aacttcgtat aatgtatgct atacgaagtt
     3781 atcgtacggg atctaattca attagagact aattcaatta gagctaattc aattaggatc
     3841 caagcttatc gatttcgaac cctcgaccgc cggagtataa atagaggcgc ttcgtctacg
     3901 gagcgacaat tcaattcaaa caagcaaagt gaacacgtcg ctaagcgaaa gctaagcaaa
     3961 taaacaagcg cagctgaaca agctaaacaa tcggctcgaa gccggtcgcc accatggcct
     4021 cctccgagga cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag ggctccgtga
     4081 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     4141 ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
     4201 cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca
     4261 agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     4321 gcgtggtgac cgtgacccag gactcctccc tccaggacgg ctccttcatc tacaaggtga
     4381 agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaag actatgggct
     4441 gggaggcgtc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca
     4501 aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg
     4561 ccaagaagcc cgtgcagctg cccggctact actacgtgga ctccaagctg gacatcacct
     4621 cccacaacga ggactacacc atcgtggagc agtacgagcg cgccgagggc cgccaccacc
     4681 tgttcctgta ggggccgcga ctctagatca taatcagcca taccacattt gtagaggttt
     4741 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     4801 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4861 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4921 tcaatgtatc ttaaccgtct gtaagcagcg aaggcccata cgataacttc gtataatgta
     4981 tgctatacga agttatgagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     5041 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5101 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5161 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5221 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5281 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5341 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5401 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5461 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5521 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5581 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5641 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5701 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5761 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5821 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5881 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5941 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6001 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6061 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6121 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6181 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6241 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6301 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6361 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6421 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6481 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6541 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6601 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6661 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6721 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6781 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6841 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6901 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6961 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7021 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7081 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7141 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
//
