LOCUS       sequence-162337-        6375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  an updated version of cLac94 (pACSA_PcptOO_YFP_PMB2__LacIWF). The 
            homologous region upstream of the inducible system was fixed to add 
            a stop codon for A1837. No difference in expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6375)
  AUTHORS   Markley AL, Begemann MB, Clarke RE, Gordon GC, Pfleger BF
  TITLE     Synthetic biology toolbox for controlling gene expression in the 
            cyanobacterium Synechococcus sp. strain PCC 7002.
  JOURNAL   ACS Synth Biol. 2015 May 15;4(5):595-603. doi: 10.1021/sb500260k. 
            Epub 2014 Sep 25.
  PUBMED    25216157
REFERENCE   2  (bases 1 to 6375)
  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"; doi: "10.1021/sb500260k";
            journalName: "ACS Synth Biol"; date: "2015-05-15- 15"; volume: "4"; 
            issue: "5"; pages: "595-603"
COMMENT     Sequence Label: pALM174
FEATURES             Location/Qualifiers
     source          1..6375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(202..221)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(203..221)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(228..245)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(228..244)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(237..259)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      385..840
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(472..491)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     682..703
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        918..1022
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1023..1091,1092..1883)
                     /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(1241..1260)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2054..2642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2543..2562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2796..2813
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3525..3544
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     protein_bind    3629..3645
                     /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)."
     CDS             3694..3723
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3736..3747
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             join(3769..3771,3772..3774,3775..4488)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3814..3835)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4075..4094)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4422..4443
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4558..4576)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4572..4619
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4717..4751
                     /label=BBa_J23110
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.33"
     CDS             4781..5863
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDFSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4800..4819)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtcccaattg tcttgcaccg tggcgatcgc cgttgcttgg gttggcgatg gtgggacggc
       61 agccaaagtt gtttgccagc gatggtggga cggcagccaa agttgtttgc cagcgatggt
      121 gggacggcag ccaaagttgt ttgccaggtg actgccgcac tcaatggtgg gacggcagcc
      181 aaagttgttt gccagaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
      241 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      301 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      361 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      421 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      481 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      541 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
      601 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
      661 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
      721 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
      781 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
      841 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca
      901 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      961 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     1021 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     1081 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     1141 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     1201 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1261 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1321 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1381 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1441 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1501 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     1561 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     1621 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     1681 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     1741 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     1801 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     1861 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     1921 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     1981 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2041 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2101 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2161 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     2221 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2281 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2341 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2401 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2461 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2521 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2581 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2641 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2701 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     2761 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     2821 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     2881 ggcacgacag gtttcccgac tggaactgcc gcactcaatg ttcaaccaga tctagtgatt
     2941 atttcggggt tagcagccga tgcgggcaac ctggtgaagc aactgcgaga attaggttac
     3001 aacggcatta ttgttggggg gaacggctta aatacttcta atattttccc cgtctgccaa
     3061 gcaaaatgtg atggggtgtt ggtggcccaa gcctacagtg ccgagttaga taatgagatt
     3121 aaccgcgcgt ttcgggacgc ctattttcaa caaaaccaaa aagagccgcc ccaatttagt
     3181 gcccaggctt ttacggcgat ccaagttttt gttgaagccc tcagcagcct cgatgaaaaa
     3241 acgcccttag aaactcttgc tctaccggac ttgcgacgac aactgcggga cgaaattttt
     3301 gcaggtacct acgtcacgcc tttgggtgaa atttccttca cagaggaagg ggaaattgtc
     3361 cagaaggaat tttttgtggc ccaaattgaa atggatgaat cgggtcaaca ggggcgtttc
     3421 gccttcattg aaacgaacta aagtcaaacc taggcctgtg tttaaaaggg ggaatcagtt
     3481 ttgtctttgg aatttactga ctcccctctg gacatctcca aacgaattgt gagcgctcac
     3541 aattcggaat tcttaacaaa aaagcaggaa taaaattaac aagatgtaac agacataagt
     3601 cccatcaccg ttgtataaat gtgtggaatt gtgagcggat aacaatttca cacaccaact
     3661 cataaagtca agtaggagat taattccatg ggccatcatc atcatcatca tcatcatcat
     3721 cacagcagcg gccatatcga aggtcgtcat atgcaggcag cagcctcgat ggtgagcaag
     3781 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     3841 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     3901 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     3961 ttcggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     4021 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     4081 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     4141 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     4201 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     4261 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     4321 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     4381 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     4441 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaacg agggcggtgc
     4501 tttggcagga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     4561 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgacg ggttttttgt
     4621 ctagatcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     4681 agagcgtcga gatcccggac accatcgaat ggcgcattta cagctagctc agtcctaggt
     4741 acaatgctag ccggaggagg gtcaattcat ggtggtgaat gtgaaaccag taacgttata
     4801 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     4861 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     4921 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     4981 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     5041 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     5101 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     5161 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     5221 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     5281 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     5341 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     5401 caatcaaatt cagccgatag cggaacggga aggcgacttt agtgccatgt ccggttttca
     5461 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     5521 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     5581 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     5641 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     5701 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     5761 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5821 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5881 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     5941 cagtcagctc cttccggttc gcgctcaagt tgatcgaatg tatgccccgg atatgaccta
     6001 gggagattct tctaaagcga gcagggtttg ccaagtggcg atcgcctcag ggaaccataa
     6061 ccaactttgt tcgaggcggc gggggttgag ctgggaacga tcgagatcta gggcgatcgc
     6121 ccggagtttt tcggctttcc ctacaagaat ttcctggtca acgtcggtgg gttgcctttg
     6181 ggccaaatcg tacatactaa tcgccaggcc cgcataaatc tccgcctgga gggctggggg
     6241 gaaatcctgg gtcgttgccg ccgctaaggc ttcataccaa cgatcattgg cggcttcgta
     6301 atctgcattg aggtaatgga caaaacccag ggccatcaaa atttcggggt cgttgggctg
     6361 ttgggctagg gccat
//
