LOCUS       sequence-398437-        5588 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty expression vector containing Cre recombinase reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5588)
  AUTHORS   Tague N, Andreani V, Fan Y, Timp W, Dunlop MJ
  TITLE     Comprehensive Screening of a Light-Inducible Split Cre Recombinase 
            with Domain Insertion Profiling.
  JOURNAL   ACS Synth Biol. 2023 Oct 3. doi: 10.1021/acssynbio.3c00328.
  PUBMED    37788288
REFERENCE   2  (bases 1 to 5588)
  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: "ACS Synth 
            Biol. 2023 Oct 3. doi: 10.1021/acssynbio.3c00328."
COMMENT     Sequence Label: pTKEI-Dest-loxP-sfGFP
FEATURES             Location/Qualifiers
     source          1..5588
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..53
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     59..78
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    complement(215..248)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      296..367
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      383..410
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    complement(419..452)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             473..1186
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1207..1278
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1294..1321
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1544..2626)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2588..2607
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2627..2704)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(2749..2754,2755..2771,2772..2778)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2786..2802
                     /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)."
     protein_bind    2898..2919
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2934..2939,2940..2957,2958..2964)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2972..2988
                     /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)."
     primer_bind     2996..3012
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2996..3012
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3008..3331
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    3025..3081
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3082..3099)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3082..3098)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3091..3113)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3307..3326)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             3429..3446
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3464..3481)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3634..3720
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3812..3839
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3945..4760)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSGPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4668..4687
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4752..4771
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4882..5470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5371..5390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaatcgg
       61 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
      121 tgcgtccggc gtagaggatc gagttatcaa aaagagtatt gaaataaagt ctaacctata
      181 ggaagattac agccatcgag agggacacgg cgaaataact tcgtatagca tacattatac
      241 gaagttatcc aggcatcaaa taaggatcca aactcgagta aggatctcca ggcatcaaat
      301 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
      361 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataccat
      421 aacttcgtat agcatacatt atacgaagtt attttaagaa ggagatatac atatgcgtaa
      481 aggcgaagag ctgttcactg gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa
      541 cggtcataag ttttccgtgc gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac
      601 gctgaagttc atctgtacta ctggtaaact gccggtacct tggccgactc tggtaacgac
      661 gctgacttat ggtgttcagt gctttgctcg ttatccggac catatgaagc agcatgactt
      721 cttcaagtcc gccatgccgg aaggctatgt gcaggaacgc acgatttcct ttaaggatga
      781 cggcacgtac aaaacgcgtg cggaagtgaa atttgaaggc gataccctgg taaaccgcat
      841 tgagctgaaa ggcattgact ttaaagaaga cggcaatatc ctgggccata agctggaata
      901 caattttaac agccacaatg tttacatcac cgccgataaa caaaaaaatg gcattaaagc
      961 gaattttaaa attcgccaca acgtggagga tggcagcgtg cagctggctg atcactacca
     1021 gcaaaacact ccaatcggtg atggtcctgt tctgctgcca gacaatcact atctgagcac
     1081 gcaaagcgtt ctgtctaaag atccgaacga gaaacgcgat catatggttc tgctggagtt
     1141 cgtaaccgca gcgggcatca cgcatggtat ggatgaactg tacaaataat agggatctcc
     1201 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     1261 ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc
     1321 gtttatacct agggatactg cggaagagcg ccacggtgat tcattctgct aaccagtaag
     1381 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc
     1441 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     1501 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     1561 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1621 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgacacgggc aacagctgat
     1681 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     1741 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     1801 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     1861 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatac
     1921 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     1981 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     2041 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     2101 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     2161 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     2221 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     2281 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     2341 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     2401 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     2461 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     2521 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     2581 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     2641 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     2701 tgtccgggat ctcgacgctc tcccttatgc gactccgatc ccgcaaattt gacaattaat
     2761 catccggctc gtataatgtg tggaattgtg agcggataac aatttcacac aaggagatat
     2821 accatgtcca gagacgatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2881 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     2941 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     3001 aacagctatg accatgatta cgccaagctt gcatgcctgc aggtcgactc tagaggatcc
     3061 ccgggtaccg agctcgaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3121 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3181 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     3241 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     3301 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     3361 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctcgtct
     3421 ctagcggtca ccatcaccat caccattaag agaagatttt cagcctgata cagattaaat
     3481 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     3541 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     3601 atccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     3661 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     3721 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     3781 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttc
     3841 tgaaaggagg aactatatcc ggatatccac aggacgggtg tggtcgccat gatcgcgtag
     3901 tcaaggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgttagaa aaactcatcg
     3961 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     4021 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     4081 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     4141 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     4201 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     4261 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgaggcga
     4321 aatacgcgat cactgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     4381 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc tagtacctgg
     4441 aatgctgttt tgccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     4501 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     4561 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     4621 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     4681 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcccaga gcaagacgtt
     4741 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     4801 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4861 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4921 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4981 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     5041 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5101 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5161 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5221 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5281 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5341 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5401 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5461 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5521 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5581 tgagtgag
//
