LOCUS       sequence-176140-        7000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP with -27 theoretical charge fused to Cre in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7000)
  TITLE     pET-(negative27)GFP-Cre
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7000)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET-(neg27)GFP-GGS9-Cre-6xHis
FEATURES             Location/Qualifiers
     source          1..7000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(34..825,826..894))
                     /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     657..676
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(895..987)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1068..1656
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1557..1576
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1810..1827
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1842..1981
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1907..1926)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2067..2089
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2083..2274)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2846..3928)
                     /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     3890..3909
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3929..4006)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4212..4231
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4319..4338
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4319..4337
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4338..4362
                     /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)."
     RBS             4377..4399
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5213..6238
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     CDS             6248..6265
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6284..6328
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(6366..6384)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6380..6427
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6464..6919
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6551..6570)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6761..6782
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
       61 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
      121 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
      181 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
      241 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
      301 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
      361 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
      421 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
      481 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
      541 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
      601 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
      661 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
      721 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
      781 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
      841 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
      901 cctttttcaa tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat
      961 ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt
     1021 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     1081 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     1141 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1201 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     1261 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1321 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1381 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1441 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     1501 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     1561 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     1621 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     1681 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     1741 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     1801 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     1861 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     1921 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     1981 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2041 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2101 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     2161 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     2221 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     2281 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     2341 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     2401 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     2461 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     2521 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     2581 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     2641 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     2701 ccgggtcctc aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga
     2761 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     2821 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2881 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     2941 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     3001 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     3061 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     3121 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     3181 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     3241 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     3301 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     3361 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     3421 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     3481 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     3541 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     3601 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     3661 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     3721 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     3781 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     3841 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     3901 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     3961 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     4021 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     4081 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     4141 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     4201 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     4261 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta
     4321 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     4381 tttaacttta agaaggagat ataccatggg tgctagcaaa ggtgaagagc tgtttgacgg
     4441 tgtagtaccg atcttagtgg aattagacgg cgacgtgaac ggtcacgaat ttagcgtgcg
     4501 cggcgagggc gaaggtgacg ctaccgaggg tgaattgacc ctgaagttta tttgcacaac
     4561 aggcgaatta cccgttccgt ggcccacctt agtgaccacc ctgacctatg gcgttcagtg
     4621 cttcagtgat tacccagatc atatgaaaca acacgatttt ttcaaatcag ccatgcctga
     4681 aggatatgtt caagagcgta caatcagctt caaggacgat ggcacctata aaacgcgtgc
     4741 ggaagtgaaa tttgaaggcg acacattagt aaaccgtatc gaactgaaag gtatcgactt
     4801 caaagaagac ggcaacattt taggccataa gctggaatat aactttaatt ctcataacgt
     4861 gtatattacg gccgataaac aggaaaacgg tatcaaggca gaatttgaaa ttcgccataa
     4921 cgtggaggac ggcagcgttc aattagcgga tcattatcaa caaaacacgc cgattggtga
     4981 tgggcctgta ctgttacctg acgatcacta cctgagcacg gagtcagccc tgagcaaaga
     5041 tccgaacgaa gaccgcgatc acatggttct gttagaattc gtgaccgctg caggcattga
     5101 tcatggaatg gacgagctgt acaagaccgg tggtagcggt ggttctggtg gttctggtgg
     5161 tagcggcggt agcggtggta gcggtggtag cggtggcagc ggcggtaccg cgagcaattt
     5221 actgaccgta caccaaaatt tgcctgcatt gccggtcgat gcaacgagtg atgaggttcg
     5281 caagaacctg atggacatgt tcagggatcg ccaggcgttt tctgagcata cctggaaaat
     5341 gcttctgtcc gtttgccggt cgtgggcggc atggtgcaag ttgaataacc ggaaatggtt
     5401 tcccgcagaa cctgaagatg ttcgcgatta tcttctatat cttcaggcgc gcggtctggc
     5461 agtaaaaact atccagcaac atttgggcca gctaaacatg cttcatcgtc ggtccgggct
     5521 gccacgacca agtgacagca atgctgtttc actggttatg cggcgtatcc gaaaagaaaa
     5581 cgttgatgcc ggtgaacgtg caaaacaggc tctagcgttc gaacgcactg atttcgacca
     5641 ggttcgttca ctcatggaaa atagcgatcg ctgccaggat atacgtaatc tggcatttct
     5701 ggggattgct tataacaccc tgttacgtat agccgaaatt gccaggatca gggttaaaga
     5761 tatctcacgt actgacggtg ggagaatgtt aatccatatt ggcagaacga aaacgctggt
     5821 tagcaccgca ggtgtagaga aggcacttag cctgggggta actaaactgg tcgagcgatg
     5881 gatttccgtc tctggtgtag ctgatgatcc gaataactac ctgttttgcc gggtcagaaa
     5941 aaatggtgtt gccgcgccat ctgccaccag ccagctatca actcgcgccc tggaagggat
     6001 ttttgaagca actcatcgat tgatttacgg cgctaaggat gactctggtc agagatacct
     6061 ggcctggtct ggacacagtg cccgtgtcgg agccgcgcga gatatggccc gcgctggagt
     6121 ttcaataccg gagatcatgc aagctggtgg ctggaccaat gtaaatattg tcatgaacta
     6181 tatccgtaac ctggatagtg aaacaggggc aatggtgcgc ctgctggaag atggcgacgg
     6241 cggatcccat caccaccacc atcactaatg actcgagtct ggtaaagaaa ccgctgctgc
     6301 gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa cctaggctgc
     6361 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     6421 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     6481 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     6541 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     6601 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     6661 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     6721 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     6781 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     6841 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     6901 atattaacgt ttacaatttc tggcggcacg atggcatgag attatcaaaa aggatcttca
     6961 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
//
